KR20080113369A - Assays to predict atherosclerosis and dysfunctional high-density lipoprotein - Google Patents

Abstract

This invention provides novel assays for the detection of dysfunctional HDL. The assays are good diagnostics and/or prognostics for atherosclerosis or other pathologies characterized by an inflammatory response. In certain embodiments the methods involve measurements of heme-related HDL-associated proteins (e.g., haptoglobin, hemopexin, etc.), and/or measurements of the relative distribution of HDL-associated proteins between HDL and the non-lipoprotein fractions of plasma/serum, and/or measurements of the ability of pro-inflammatory HDL to consume nitric oxide, and/or measurement of the ability of HDL to inhibit LDL aggregation. ® KIPO & WIPO 2009

Description

Assay to predict atherosclerosis and dysfunctional high density lipoprotein {ASSAYS TO PREDICT ATHEROSCLEROSIS AND DYSFUNCTIONAL HIGH-DENSITY LIPOPROTEIN}

Cross Reference to Related Application

This application claims the benefits and priorities of USSN 60 / 843,213, filed September 7, 2006 and USSN 60 / 772,429, filed February 10, 2006, all of which are hereby incorporated by reference in their entirety for all purposes. do.

A statement of the rights of the invention, made under federally sponsored research and development

This study was partly sponsored by grant numbers 1RO1HL7 1776 and HL 30658 from the National Institutes of Health. The US government has certain rights in this invention.

The present invention relates to the diagnosis of atherosclerosis. In particular, the present invention provides an improved assay for the detection of dysfunctional HDL.

Atherosclerosis, a chronic inflammatory disease of large and medium arteries, is the leading cause of morbidity and mortality in Western countries. The introduction of statins reduced mortality by one third. However, despite statin treatment, two-thirds mortality from the disease continues.

Oxidation of low density lipoproteins (LDL) is a major factor in human atherosclerosis (Witztum and Steinberg (2001) Trends Cardiovasc . Med ., 11: 93-102; Witztum and Steinberg (1991) J. Clin . Invest ., 88: 1785-1792). The capture and oxidation of LDL in the sub-endothelial space, and subsequent interaction between epithelial cells and monocytes, are a key process in the onset of atherosclerotic lesion development (Navab et al. (1996) Arterioscler. Thromb . Vasc . Biol ., 16: 831-842; Berliner et al. (1995) Circulation 91: 2488-2496). Minimal modification / oxidation-LDL (MM-LDL) contains biologically active molecules that can induce epithelial cells to produce inflammatory factors such as chemokines, adhesion molecules, and growth factors. These inflammatory molecules promote the recruitment and adhesion of monocytes to epithelial cells (Berliner et al. (1995) Circulation 91: 2488-2496). Several biologically active oxidized phospholipids have been identified in atherosclerotic lesions of MM-LDL and animal models (Watson et al . (1995) J. Clin . Invest ., 95: 774-782 ; Watson et al . (1995) J. Clin . Invest ., 96: 2882-2891; Watson et al . (1997) J. Biol . Chem ., 272: 13597-13607; Watson et al . (1999) J. Biol . Chem ., 274: 24787-24798; Leitinger et al . (1999) Arterioscler. Thromb . Vasc . Biol . , 19: 1291-1298; Subbanagounder et al. (2000) Free Radic . Biol . Med . , 28: 1751-1761). Oxidized-L-α-1-palmitoyl-2-arachidonyl- sn -glycero-3-phosphocholine (ox-PAPC) and three of its components, 1-palmitoyl-2- (5- glycerophosphate 3-phosphocholine (POVPC), 1-palmitoyl-2-yl glue Taro-yl) oxo balreo-sn sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl- 2- (5,6-epoxy-isopropenyl Stan E ₂₎ - sn - glycero-3-phosphocholine in (PEIPC) (Watson et al. (1999) J. Biol Chem, 274:.. 24787-24798; Leitinger et al. ( Proc . Natl . Acad . Sci . USA (1999) 96: 12010-12015; Subbanagounder et al. (2000) Arterioscler . Thromb . Vasc . Biol ., 20: 2248-2254) allow monocytes to bind to epithelial cells, It plays a major role in the activation of epithelial cells by MM-LDL. Following the discovery of these molecules, a series of other oxidized phospholipids formed by oxidation of unsaturated fatty acids at the sn-2 position of the phospholipids with similar biological activity have been identified (Berliner and Watson (2005) N. Engl . J ) . . Med, 353:. 9-11) .

The inverse relationship between HDL and the risk of atherosclerosis is well established. A single explanation for the anti-atherogenic role of HDL does not appear to exist, but it is clear that the functional phase of HDL, which depends largely on its protein components, is an important determinant of coronary artery disease (CHD). Navab et al. (2001) Arterioscler . Thromb . Vasc . Biol., 21: 481-488). Paraoxonase 1 (PON1), lecithin-cholesterol acyltransferase (LCAT), platelet-activating factor acetyl hydrolase (PAF-AH), proteinase (elastase-like), phospholipase D, albumin , apoJ and apoA-I are proteins in HDL with anti-atherogenic properties that can prevent MM-LDL formation. HDL has been shown to play a role in preventing LDL oxidation. HDL inhibited the moderate oxidation of LDL and, as a result, inhibited the production of MCP-1, a potent monocyte chemoattractant by human arterial wall cells.

HDL may exist as anti-inflammatory or pro-inflammatory molecules, depending on context and environment. In rabbits and humans, an acute phase reaction (APR) can convert HDL from an anti-inflammatory form to a pro-inflammatory form, ie, HDL loosens its ability to protect against LDL-induced inflammation, and its precursor In inflammatory conditions, HDL actually promotes LDL-induced inflammation. Without being bound by a particular theory, under basal conditions, HDL plays an anti-inflammatory role, but during APR loss of anti-oxidant enzyme activity, impairment of apoA-I, and displacement and / or displacement of proteins associated with HDL It is believed that there is production of pro-inflammatory HDL by exchange. For example, in mice genetically susceptible to atherosclerosis (but not in mice that are genetically resistant to atherosclerosis), atheromatous diets have been shown to convert HDL from anti-inflammatory to pro-inflammatory (Navab et al. 1997) J. Clin . Invest ., 99: 2005-2019). Subsequently, several studies have shown the presence and nature of pro-inflammatory HDL in animal models (Castellani et al . (1997) J. Clin . Invest ., 100: 464-474, reviewed by Navab et al. (2005) Ann . Med, 37:. 173-178) .

HDL from C57BL / 6J mice (a species genetically susceptible to dietary-induced atherosclerosis) was anti-inflammatory in mice in a chow diet, but pro-inflammatory when the mice were in an atheromatous diet. Shih et al. (1996) J. Clin. Invest ., 97: 1630-1639. In contrast, HDL from atherosclerosis-resistant C3H / HeJ (C3H) mice was anti-inflammatory regardless of whether the mice were on a normal or atheromatous diet (see above). All mouse models that have developed atherosclerosis with macrophage-rich lesions studied so far have pro-inflammatory HDL. These include: C57BL / 6J mice provided with an atheromatous diet (see above), transgenic mice overexpressing apoA-II provided with a normal diet (Castellani et al . (1997) J. Clin . Invest . , 100: 464-474), PON1 deficient (null) mice fed with a fed atherogenic diet (supra), apoE deficient mice (Navab et al . (1997) J. Clin . Invest ., 99: 2005-2019), ApoE deficient and PON1 deficient combination mice given normal diet (Castellani et al . (1997) J. Clin . Invest ., 100: 464-474), LDL receptor (LDLR) deficient mice fed a high fat diet (Navab et al. (1997) J Clin. Invest ., 99: 2005-2019), and transgenic mice overexpressing sPLA ₂ (Leitinger et al. (1999) Arterioscler . Thromb . Vasc . Biol . , 19: 1291-1298). In genetically distinct mouse models, all of which are hyperlipidemic but none develop atherosclerosis, HDL has been shown to be anti-inflammatory (Navab et al. (2005) Ann. Med ., 37: 173-178). On the other hand, seven other mouse models that developed atherosclerosis, all characterized by macrophage-rich lesions, had pro-inflammatory HDL (supra), which showed that HDL function had anti- or pro-inflammatory properties of HDL. It is a more sensitive marker for the presence or absence of atherosclerosis rather than cholesterol levels. The quality and function of HDL has been an attractive target for emerging new therapies (Castellani et al . (1997) J. Clin . Invest ., 100: 464-474; Navab et al . (2005) Ann . Med ., 37: 173-178; Ridker (2002) Circulation , 105: 2-4; Ansell et al. (2003) Circulation , 108: 2751-2756).

Although many protein and enzyme activities have been associated with HDL, little is known about which specific protein profile is associated with pro-inflammatory HDL.

Summary of the Invention

Determining the quality and function of HDL is important for improving detection of individuals at risk of atherosclerotic events and for targeting new and novel therapies. Current testing for dysfunctional HDL is a measure of the various parameters and components of the HDL, which are relatively cumbersome and expensive.

The present invention provides a new simple assay for predicting dysfunctional HDL.

In certain embodiments the invention relates to the identification of protein profiles that distinguish pro-inflammatory HDL from normal / anti-inflammatory HDL. This provides a biomarker for the early detection of atherosclerosis, the presence of other pathologies characterized by an inflammatory response and / or predisposition, thus providing a new strategy for therapeutic intervention.

In various embodiments the assays measure the heme-associated HDL-associated protein (eg, haptoglobin, hemopexin, etc.), and / or between the non-lipoprotein fraction of plasma / serum and HDL. Determination of the relative distribution of the HDL-associated protein, and / or determination of the nitric oxide consumption capacity of the pro-inflammatory HDL, and / or determination of the ability to inhibit LDL aggregation of the HDL.

In certain embodiments the invention provides a method for detecting or quantifying pro-inflammatory HDL in a mammal. The method typically involves the following steps: providing a sample comprising HDL as a biological sample from a mammal; And detecting at least two proteins, at least three, or at least four, or at least five, at least six, at least seven, or at least eight different proteins associated with HDL, wherein the proteins are m / z Protein with ratio of about 9.3, protein with m / z ratio of about 14.9, protein with m / z ratio of about 15.6, protein with m / z ratio of about 15.8, protein with m / z ratio of about 16.2, protein with m / z ratio of about 16.5 , a protein having an m / z ratio of about 18.6, and a protein having an m / z ratio of about 19.5, and detecting at least two proteins associated with the HDL indicates that the HDL is a pro-inflammatory HDL. In certain embodiments the mammal is a non-human mammal or human (eg, Human with atherosclerosis, or at risk of atherosclerosis, or with another pathology characterized by an inflammatory response, or diagnosed with a risk of such pathologies).

In certain embodiments, a method of detecting or quantifying pro-inflammatory HDL in a mammal is provided. The method typically includes measuring the level of heme-containing and / or heme-binding protein (s) associated with HDL from the mammal, wherein the heme-containing and / or heme-binding protein ( Increased levels compared to the levels of heme-containing and / or heme-binding proteins found in normal anti-inflammatory HDLs indicate that the HDL is a pro-inflammatory HDL. In certain embodiments the heme-containing and / or heme-binding protein comprises one or more proteins selected from the group consisting of hemoglobin, haptoglobin, hemopexin, transferrin, soluble CD163, and myeloperoxidase. . In certain embodiments the method involves measuring the amount of hemoglobin associated with HDL and measuring the amount of haptoglobin associated with HDL. In certain embodiments the method comprises producing a product of hemoglobin and haptoglobin associated with HDL. In certain embodiments the method involves measuring the level of said same heme-containing and / or heme-binding proteins in a non-lipoprotein fraction of plasma, wherein heme-containing and / or in HDL Or an increase in the ratio of heme-containing and / or heme-binding protein in the non-lipoprotein fraction of heme-binding protein to plasma is increased compared to the ratio found in subjects with normal anti-inflammatory HDL. It indicates that HDL from the animal is pro-inflammatory HDL.

Also provided are methods for detecting or quantifying pro-inflammatory HDL in a mammal. In various embodiments, the method involves the following steps: measuring the level of one or more heme-containing and / or heme-binding protein (s) associated with HDL from the mammal; Measuring the level of one or more of the same heme-containing and / or heme-binding protein (s) in the plasma (or serum) of the mammal, wherein HDL heme-containing and / or heme-binding protein versus plasma heme- The ratio of containing and / or heme-binding protein to greater than 1 indicates that the HDL from the mammal is pro-inflammatory HDL. In certain embodiments the heme-containing and / or heme-binding protein comprises one or more proteins selected from the group consisting of hemoglobin, haptoglobin, hemopexin, transferrin, soluble CD163, and myeloperoxidase. In certain embodiments the heme-containing and / or heme-binding protein comprises haptoglobin and hemopexin. In certain embodiments HDL heme-containing and / or heme-binding protein to plasma heme-containing and / or heme-binding protein in at least 2, at least 3, at least 4, at least 5 or at least 6 of said proteins. A ratio of greater than 1 indicates that the HDL from the mammal is pro-inflammatory HDL. In certain embodiments the HDL heme-containing and / or heme-binding protein versus plasma heme-containing and / or heme-binding protein in one, two, three, four, five, or six of said proteins. The ratio of greater than the value selected from the group consisting of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 indicates that the HDL from the mammal is pro-inflammatory HDL.

Also provided are methods for detecting or quantifying pro-inflammatory HDL in a mammal. The methods typically involve determining the level of heme associated with HDL from the mammal, where the level of heme associated with HDL is increased compared to the level of heme associated with protective HDL. It indicates that the HDL is a pro-inflammatory HDL. In certain embodiments the increased level is statistically significant at a confidence level of at least 90%, 95%, 98% or 99%.

In certain embodiments methods are provided for detecting or quantifying pro-inflammatory HDL in a mammal, wherein the methods typically involve the following steps: determining the iron content of the HDL from the mammal, The level of iron content of the HDL increased compared to the iron content of normal anti-inflammatory HDL indicates that the HDL from the mammal is a pro-inflammatory HDL. In certain embodiments the increased level is statistically significant at a confidence level of at least 90%, 95%, 98% or 99%.

In various embodiments, methods are provided for detecting or quantifying pro-inflammatory HDL in a mammal. The methods typically involve determining the level of iron-containing proteins associated with HDL from the mammal, where the level of iron-containing proteins associated with the HDL is associated with normal anti-inflammatory HDL. Increased relative to the level of iron-containing proteins present indicates that HDL from the mammal is pro-inflammatory HDL. In certain embodiments the increased level is statistically significant at a confidence level of at least 90%, 95%, 98% or 99%.

Also provided are methods for detecting or quantifying pro-inflammatory HDL using a nitrogen monoxide assay. The methods typically involve the nitrogen monoxide consumption capacity of the HDL, wherein the increase in the nitrogen monoxide consumption capacity of the HDL compared to the nitrogen monoxide consumption capacity of the normal anti-inflammatory HLD is the presence, amount or activity of the pro-inflammatory HDL. Indicates. In certain embodiments the nitrogen monoxide is chemically produced. In certain embodiments the nitrogen monoxide is measured by an electronic signal.

Also provided are aggregation assays that detect or quantify pro-inflammatory HDL in mammals. These assays typically involve contacting HDL from the mammal with LDL and measuring aggregation of the LDL, wherein the level of LDL aggregation is in aggregation of LDL in contact with normal anti-inflammatory HDL. Increased compared to indicates that HDL from the mammal is pro-inflammatory HDL. In certain embodiments the aggregation is determined by spectroscopic measurement of LDL aggregation rate. In certain embodiments the aggregation is determined using an albumin depletion column.

In certain embodiments methods are provided for assaying the presence or predisposition of pathology characterized by an inflammatory response in a mammal. The methods typically involve performing any one or more of the assays described herein, wherein the positive test result is a labeling factor for the presence or predisposition of the pathology. In various embodiments, the pathology is atherosclerosis, stroke, leprosy, tuberculosis, systemic lupus erythematosus, rheumatic polymyalgia, nodular polyarteritis, scleroderma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, Alzheimer's disease, chronic renal failure , Diabetes, diabetic retinopathy, diabetic kidney disease, transplant rejection, graft atherosclerosis, reperfusion ischemia, adult respiratory syndrome, congestive heart failure, glomerulitis, metabolic syndrome, multiple sclerosis, sepsis syndrome, sickle cell disease, vascular disease Dementia, Crohn's disease, endothelial dysfunction, arterial dysfunction, AIDS, rheumatoid polymyalgia, nodular polyarteritis, scleroderma, idiopathic pulmonary fibrosis, coronary artery calcification, calcified aortic stenosis, osteoporosis, bacterial infections, viral infections, fungal infections, Autoimmune disorders, and rheumatoid arthritis.

In certain embodiments, the assays described herein can be used in non-human mammals or humans (eg, For samples from humans having atherosclerosis, or at risk of atherosclerosis, or with another pathology characterized by an inflammatory response, or a human diagnosed with a risk of such pathology. Is performed.

In certain embodiments, proteins detected and / or quantified in the assays described herein are immunoassay (eg, ELISA) to detect and / or quantify.

Also provided are methods of treating a human or non-human mammal with a pathology characterized by an inflammatory response. Such methods typically comprise performing any one or more of the assays described herein and more aggressive therapies (eg, statins, and / or US Pat. Nos. 7,166,578, 7,148,197, 7,144,862, Administration of a therapeutic agent as described in 6,933,279, 6,930,085, and 6,664,230, the patents of which are incorporated herein by reference for all purposes.

Justice

The following abbreviations may be used herein: APR, acute phase reaction; CAD, coronary artery disease; HDL-C, HDL cholesterol; MM-LDL, minimal strain / oxidation-LDL; PON, paraoxonases; ApoA1, Apolipoprotein A1; apoE, apolipoprotein E; CM10, weak cation chip; CVD, cardiovascular disease; CHD, coronary artery disease; FPLC, high speed protein liquid chromatography; Hb, hemoglobin; Hb-alpha, hemoglobin alpha chains; Hb-beta, hemoglobin beta chains; Hp, haptoglobin; Hx, hemopexin; HDL, high density lipoproteins; HPLC, high performance liquid chromatography; LDL, low density lipoproteins; LDLR, LDL receptor; metHb, methemoglobin; NP20 chip, normal phase chip; oxy Hb, oxyhemoglobin; PON, paraoxonases; pHDL, post HDL fraction; Q10 chip, strong anion-exchange chip; RBC, erythrocytes; SELDI, surface-enhanced laser desorption / ionization; SELDI-TOF-MS, surface-enhanced laser desorption / ionization time-of-flight mass spectrometer; micro-liquid chromatography using μLC-MSMS, tandem mass spectrometry; VLDL, ultra low density lipoprotein.

"Immunoassay" is an assay that uses an antibody that specifically binds to an analyte (eg, haptoglobin and / or hemopexin). The immunoassay is characterized by isolating, targeting and / or quantifying the analyte using the specific binding properties of a particular antibody.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers.

The term “improvement” when used in connection with “ameliorating the symptoms of one or more atherosclerosis” refers to the reduction, prevention or elimination of one or more symptoms peculiar to atherosclerosis and / or related pathology. Such reductions include, but are not limited to, reduction or elimination of oxidized phospholipids, reduction of atherosclerotic plaque formation and rupture, reduction of clinical events such as heart attack, angina, or stroke, hypotension, Inflammatory protein biosynthesis, decreased plasma cholesterol, and the like.

The term "low density lipoprotein" or "LDL" is defined in accordance with conventional practice of those skilled in the art. In general, LDL refers to a lipid-protein complex that is found in the density range of d = 1.019 to d = 1.063 when isolated by ultracentrifugation.

The term "high density lipoprotein" or "HDL" is defined in accordance with conventional practice of those skilled in the art. Generally "HDL" refers to a lipid-protein complex that is found in a density range of d = 1.063 to d = 1.21 when isolated by ultracentrifugation.

The term "Group I HDL" or "protective HDL" or "normal anti-inflammatory HDL" refers to a high density lipoprotein that reduces oxidized lipids (such as in low density lipoproteins) or protects lipids from oxidation by oxidizing agents or Components thereof (such as apo AI, paraoxonases, platelet activating factor acetyl hydrolases, and the like).

The term “dysfunctional HDL” provides reduced activity or no activity in protecting lipids from oxidation or in restoring (eg, reducing) oxidized lipids, thereby preventing the inflammatory ending of these oxidized lipids. It refers to HDL, which may or may not be substantially preventable.

The term "HDL component" refers to a component (such as a molecule) comprising a high density lipoprotein (HDL). Assays for HDL that protect or repair lipids from oxidation (such as reducing oxidized lipids) also include components of HDL that exhibit this activity (eg , apo AI, paraoxonases, platelet activating factor acetyl hydrolases, etc.). Test for).

"Hemopexin" or "haptoglobin" is a surrogate marker for hemopexin or haptoglobin, such as full length native hemopexin or haptoglobin, or hemopexin or haptoglobin fragments, isozymes, etc., respectively. Its detection / quantification refers to a marker that provides a measure of the amount / concentration of the full-length molecule.

The term “antibody” refers to a polypeptide that is substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, that recognize and specifically bind the analyte (antigen). The recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, and countless immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Typical immunoglobulin (antibody) structural units consist of tetramers. Each tetramer consists of two identical polypeptide chain pairs, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50-70 kD). The N-terminus of each chain is characterized by a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to these light and heavy chains, respectively.

Antibodies exist, for example, as intact immunoglobulins or as fully characterized fragments generated by cleavage by a number of various peptidases. That is, for example, pepsin cleaves antibodies under disulfide linkages within their hinge regions to produce F (ab) ' ₂ , a dimer of Fab, which itself is linked to V _H -C _H 1 by disulfide bonds. Light chain. The F (ab) ' ₂ is reduced under moderate conditions to break the disulfide linkage in the hinge region, converting the F (ab)' ₂ dimer into a Fab 'monomer. The Fab 'monomer is essentially a Fab with part of the hinge region (Fundamental Immunology, 3rd edition, WE Paul, ed., Raven Press, NY 1993). Various antibody fragments are defined in terms of cleavage of intact antibodies, but one of ordinary skill in the art will recognize that such fragments can be newly synthesized chemically or using recombinant DNA methodology. That is, as used herein, the term antibody also refers to antibody fragments produced by modification of whole antibodies, those newly synthesized using recombinant DNA methodology (eg single chain Fv), and display libraries (eg phage display library). Include what is found.

The phrase “normal healthy control” refers to an individual or population of individuals of approximately the same age and same sex that are asymptomatic or have negative test results for the condition / pathology.

The phrase “detecting the level of haptoglobin and / or hemopexin and / or other proteins in the blood or blood fraction” refers to haptoglobin and / or hemopexin in the blood, blood fraction, or sample derived from the blood or blood fraction. And / or detection and / or quantification of other proteins. The detection may be direct detection of intact haptoglobin and / or hemopexin, and / or other proteins and / or detection of haptoglobin and / or hemopexin and / or other protein fragments, detection of isoforms, and / or May involve the detection of various other surrogate markers for the protein (s) of interest.

The phrase “providing a biological sample from a mammal” means either taking the sample directly (eg , a blood sample) or obtaining or providing a biological sample taken from a mammal by a third party.

Brief description of the drawings

1 shows western analysis of HDL associated proteins. This Western analysis showed that the association of HDL with hemoglobin (Hb), haptoglobin (Hp), and hemopexin (Hx) in C57BL / 6J mice fed an atheromatous diet (A) compared to normal diet (C). Proved to increase by more than 10 times. As shown in this figure, these proteins did not associate with VLDL or LDL.

2 shows that ApoE deficient mouse HDL is pro-inflammatory on normal diet and converts to anti-inflammatory after oral D-4F treatment. Nine-month-old female apoE deficient mice (n = 4 per group) on a normal diet were treated with apolipoprotein AI mimetic peptide D-4F (per mL of drinking water) before treatment (day 0) and for the number of days shown on the X-axis. Bleeding after treatment with 50 μg). LDL + DCF fluorescence inhibition ability of apoE deficient mouse HDL was measured as a measure of inflammatory properties. The data indicate that apoE deficient mouse HDL was pro-inflammatory prior to treatment (ie, addition of day 0 apoE deficient HDL increased the fluorescence above that induced by LDL alone) but after treatment with D-4F (first Day 21) has become anti-inflammatory.

3 shows that the serum concentration of hemopexin is lowered after D-4F treatment. Serum concentrations of hemopexin in the mice described in FIG. 2 were measured by ELISA.

4 shows that the serum concentration of haptoglobin is lowered after D-4F treatment. Serum concentration of haptoglobin in the mouse described in Figure 2 was measured by ELISA.

5 shows that D-4F treatment reduces haptoglobin (Hp) and hemopexin (Hx) in HDL of apoE deficient mice. HDL was isolated from the mice described in FIG. 2 and the contents of Hp, Hx, and apolipoprotein A-I (ApoA-I) were measured.

FIG. 6 shows an increase in non-RBC hemoglobin on the original PAGE gel with molecular weight similar to the hemoglobin contained in RBC as a result of D-4F treatment. Serum, HDL and red blood cells (RBC) from each of the four mice described in FIG. 2 before treatment (day 0) and after treatment 21 days (day 21) were run on an original PAGE (4-15%) gel. Hemoglobin was analyzed by Western analysis. The data shows that all hemoglobin in the serums and HDL-supernatants (containing HDL and non-lipoprotein fractions) had a significantly higher apparent molecular weight than hemoglobin from RBCs that had dissolved prior to treatment. It is shown to run on a gel. However, 21 days after treatment with D-4F, a significant amount of hemoglobin in the serums and HDL-supernatants was run on a gel having an apparent molecular weight similar to hemoglobin from dissolved RBCs.

Figure 7 shows an increase in both alpha and beta chains of hemoglobin in wild type C57BL / 6J mice fed with an atheromatous diet. The figure also shows an increase in the content of both the α and β chains of hemoglobin in apoE deficient mice on a normal diet and both chains decreased by D-4F treatment as described in FIG. 2.

Figure 8 shows the contents of oxy-hemoglobin and met-hemoglobin in HDL in wild-type C57BL / 6J mice fed with a normal diet for 7 days (D7C) or with atherogenic diets for 7 days (D7A) or for 15 weeks (W15A). Indicates that was measured. Oxy-hemoglobin and met-hemoglobin content was also measured in apoE deficient mice (apoE) fed a normal diet. As shown in this figure, the majority of HDL-associated hemoglobin in C57BL / 6J mice on atheromatous diet and apoE deficient mice on normal diet was oxy-hemoglobin.

9 shows that normal diet did not consume the chemically generated nitric oxide (NO donor) (measured as current (pA)) of HDL addition from wild-type C57BL / 6J mice (D7C) fed for 7 days. .

FIG. 10 shows that the addition of oxy-hemoglobin (HbO ₂ ) caused a transient sharp decrease in the decomposition curve of chemically generated nitrogen monoxide (NO donor) (measured in current (pA)). The addition of phosphate buffered saline (PBS) did not alter the original degradation curve. In contrast, the addition of HDL (D7A HDL-Hb) from wild-type C57BL / 6J mice fed with an atherogenic diet for 7 days resulted in a drastic and dramatic decrease in the degradation curve, which pro-inflammatory HDL caused It shows the rapid consumption of nitrogen monoxide.

FIG. 11 shows that pro-inflammatory HDL (HDL from wild-type C57BL / 6J mice fed with an atheromatous diet for 7 days; D7A HDL) caused a drastic and dramatic decrease in nitric oxide degradation curves. It indicates that inflammatory HDL consumed nitric oxide rapidly. However, after treatment of the HDL with K ₃ Fe (CN) ₆ which converted oxy-hemoglobin (HDL-HbO ₂ ) in HDL to met-hemoglobin (HDL-metHb), the addition of HDL significantly altered the degradation curve. This did not indicate that nitrogen monoxide was not consumed.

12 shows that a normal diet is fed for 7 days (left top panel), or 15 weeks (right top panel), or an atherogenic diet is fed for 7 days (left bottom panel) or 15 weeks (right bottom panel). Western analysis of the sera of wild-type C57BL / 6J mice is used to show two-dimensional gels stained for hemoglobin. The column at the left end of each panel indicates the location of hemoglobin in lysed red blood cells (RBCs) from each condition.

FIG. 13 shows the bottom panels of FIG. 12 in which an atheromatous diet was supplied to wild type C57BL / 6J mice for 7 days (D7A) or for 15 weeks (W15A), with hemoglobin staining being light blue Appears as (grayed out).

Figure 14. The western blot shown in Figure 13 was stripped off and re- probed with antibodies to haptoglobin. The resulting images (magenta) were superimposed on the images of FIG. 13. Dark blue areas represent areas where both hemoglobin and haptoglobin co-localize.

15 . The western blot shown in FIG. 14 was stripped off and re- probed with an antibody against hemopexin. The resulting images (yellow) were superimposed on the images of FIG. Very dark images show areas where hemoglobin, haptoglobin, and hemopexin coexist.

FIG. 16 shows that pro-inflammatory HDL does not inhibit LDL aggregation while anti-inflammatory HDL inhibits. Induced by phospholipase C (PLC) for HDL from 4 subjects (CHD patients) and 4 healthy volunteers (normal) with pro-inflammatory HDL and coronary artery disease or equivalent by NCEP ATP III criteria The ability to inhibit LDL aggregation was tested. Values for the positive control (LDL + PLC) without the addition of HDL are shown as red lines. Values for the negative control (LDL alone) are indicated by the bottom blue line. The data indicate that HDL from four CHD patients could not inhibit PLC induced LDL aggregation, while HDL from four healthy volunteers (normal) significantly inhibited PLC induced LDL aggregation.

Figure 17 shows that pro-inflammatory apoE deficient HDL does not inhibit phospholipase C (PLC) induced LDL aggregation before treatment, but after 21 days of oral D-4F treatment, the HDL becomes anti-inflammatory and PLC-induced LDL. Show inhibition of aggregation. The ability to inhibit PLC-induced LDL aggregation was tested for apoE deficient HDL before and after treatment with oral D-4F as described in FIG. 2. As shown in FIG. 2, the HDL was pro-inflammatory prior to treatment. As shown in FIG. 17, prior to treatment the apoE deficient HDL did not inhibit PLC-induced LDL aggregation. However, after 21 days of treatment, the HDL became anti-inflammatory (FIG. 2) and, as shown in FIG. 17, significantly inhibited PLC-induced LDL aggregation. Values for positive control (LDL + PLC (HDL free)) are shown and negative controls i) LDL + PLC with HDL (normal mouse HDL) from normal C57BL / 6J mice on normal diet and ii The values for LDL (LDL alone) of the PLC member are likewise shown.

18 shows cholesterol profile of C57BL / 6J mouse serum samples after FPLC fractionation. 500 μL pooled serum from mice (n = 8) was fractionated by FPLC. The first 10 1 mL fractions were discarded and the cholesterol content was analyzed for each next 1 mL. C-7-day normal diet, A-7-day atherogenic diet, CC-21-day normal diet, AC-7-day atherogenic diet, 14-day normal diet.

19 shows the inflammatory properties of HDL from C57BL / 6J mouse serum. HDL was isolated with HDL reagent and analyzed for active oxygen species content (fluorescence intensity), paroxase (PON) activity assay, cholesterol efflux assay (% cholesterol efflux), and cholesterol content (HDL-cholesterol). C-7-day normal diet, A-7-day atherogenic diet, AC-7-day normal diet, 14-day normal diet. ^* Represents p <0.01.

20 shows increased levels of m / z 14.9k (hemoglobin alpha chain) and m / z 15.6k (hemoglobin beta chain) in four atherosclerotic mouse models. C57BL / 6J mice given an atheromatous diet (D7 = 7 days, W15 = 15 weeks) or a western diet (WD) for 10 days, or LDLR deficient mice given Western diets for 8 weeks (LDLR Or serum samples from 12-week-old apoE deficient mice (apoE) given normal diets were analyzed by SELDI using a Q10 (pI <7) ProteinChip array. The two SELDI peaks of interest (m / z 14.9k and m / z 15.6k) in atheromatous serum were those of the serum from the corresponding mouse control group provided with a normal diet or of the same week given a normal diet for apoE deficient mice. Compared with C57BL / 6J mice, the resulting intensities were statistically analyzed. Data presented are mean increase multiples at each peak. The data presented are all statistically significant with p values <0.05. It should be noted that the assay is for proteins captured on the Q10 protein-chip that are both hydrophobic and capture proteins and complexes with pI <7.0. Hemoglobin analyzed by this method represents HDL-associated hemoglobin.

FIG. 21 shows the determination of pi for peaks representing m / z 14.9k (hemoglobin alpha chain) and m / z 15.6k (hemoglobin beta chain). Individual serum samples from C57BL / 6J mice (n = 8) given normal diet (C) or atheromatous diet (A) for 7 days (D7) or 15 weeks (W15) were buffered at different pH as shown. And desalted and fractionated with an anion exchange spin column. Eluted fractions were analyzed by SELDI using either cation exchange (CM 10: pi> 4) or anion exchange (Q10: pi <4) ProteinChip arrays. Relative intensities at 14.9k and 15.6k are shown.

22, 22B and 22C compare non-RBC hemoglobin and RBC hemoglobin, and compare the distribution of non-RBC hemoglobin in lipoprotein and non-lipoprotein fractions of serum. Serum and lipoproteins were loaded on 15% SDS-PAGE and immunoblotted against hemoglobin. As a standard for hemoglobin, RBC lysates obtained from mice of normal diet were loaded. 22A: Serum samples from C57BL / 6J mice given normal diet (C) or atheromatous diet (A) for 7 days (D7) or 15 weeks (W15) (n = 8); 22B: VLDL, LDL, HDL and post HDL (pHDL) FPLC fractions from pooled serum samples of D7 and W15 groups. FIG. 22C: Cholesterol (OD 490) and heme (OD 410) of individual FPLC fractions containing HDL and pHDL regions (fractions 25-40) from D7-C and D7-A serum samples were analyzed. 22A demonstrates that there was no significant difference in the amount of total non-RBC hemoglobin in mice fed with the normal or atheromatous diet for 7 or 15 days. The figure also shows that the molecular weights of non-RBC hemoglobin and hemoglobin from RBC dissolved on SDS PAGE gels were the same. FIG. 22B shows the conversion of non-lipoprotein fractions (pHDL) to HDL fractions in non-RBC hemoglobin by feeding an atherogenic diet for 7 days or 15 weeks. FIG. 22C corroborates the data of FIG. 24B showing that there was little hemoglobin associated with HDL after 7 days of normal diet while significant hemoglobin was present in the HDL fractions after 7 days of atheromatous diet. do.

23A and 23B show that non-RBC hemoglobin in atheromatous serum and HDL fractions in atheromatous diet has unique properties. Degree 23A: Pooled serum samples from C57BL / 6J mice (n = 8) fed a normal diet (C) or atheromatous diet (A), fractionated by FPLC, normal phase (NP20) or anion exchange (Q10) pI <7) SELDI analysis using a ProteinChip array. Relative intensities at 14.9k (Hb-alpha) and 15.6k (Hb-beta) are shown. Degree 23B: Anion exchange column fractions representing pH7.0 and pH4.0 described in FIG. 2 were loaded on 15% SDS-PAGE and immunoblotted for hemoglobin. The altered properties of non-RBC hemoglobin on atheromatous diets, as determined in these systems, are consistent with the association of hemoglobin with HDL and other HDL proteins.

24 shows that hemoglobin in atheromatous serum has unique pI values. Serum samples (n = 8) were collected from C57BL / 6J mice given normal diet (C) or atheromatous diet (A) for 7 days (D7) or 15 weeks (W15) prior to use. RBCs from D7 mice were isolated by serum gradient, washed and lysed. Serum samples were fractionated by FPLC and HDL fractions from D7 mice were collected. Intravitreal serum, HDL and RBC lysates were loaded on IEF gels (pH 3-10) and immunoblotted for hemoglobin. RBC lysates (left lanes) from mice of normal diet were loaded as a standard for hemoglobin. This figure shows that the properties of RBC hemoglobin were not different from mice of normal or atheromatous diet. The figure also shows that HDL associated hemoglobin was absent from mice fed a normal diet, but HDL associated hemoglobin was present from mice fed an atheromatous diet and its pi was clearly different from RBC hemoglobin.

FIG. 25 shows that non-RBC hemoglobin associates with HDL fractions of atheromatous diet (left panel) and that non-RBC hemoglobin migrates similarly to RBC hemoglobin after 15 weeks of feeding the atheromatous diet. Shows disappearing (right panel). From C7BL / 6J mice (n = 8) or mixed serum samples (right panel) from normal diet (C) or atheromatous diet (A) fed 7 days (D7) or 15 weeks (W15) Infusion fractions of the HDL of the (left panel) were loaded on the original-PAGE and immunoblotted for hemoglobin. RBC lysates from mice of normal diet were also loaded onto the gel (far left of the left panel).

26A and 26B show the results of spectroscopic measurements on Hb in HDL. The amount and morphology of Hb was determined from the mixed FPLC fractions containing HDL using a Beckman DU 640 spectrophotometer. Spectra of all samples and pure species were scanned at 380-700 nm (FIG. 26A). To observe the conversion of oxyHb to metHb, a slow time-release NO donor, spermine NONOate, was added to the samples (FIG. 26B, representative). The "basis spectrum" of a group of pure species was fitted to the measured spectrum using linear regression to deconvolution the concentrations of oxyHb and metHb (Vaughn). (2000) J. Biol . Chem ., 275: 2342-2348).

27 compares hemoglobin in HDL from 10 healthy volunteers with 10 patients with coronary artery disease (CHD) or equivalent disease as defined in the NCEP ATPIII guidelines. The left panel shows immunoblotting against hemoglobin as the original PAGE gel of HDL fractions from plasma collected from 10 healthy volunteers (left lane) or 10 CHD patients (right lane). The right panel shows the same analysis after dissolving RBC and adding lysate to the plasma before isolation of the HDL fractions. The results indicate that there were much more hemoglobin associated with HDL in these patients than in healthy volunteers (left panel). The figure also demonstrates that adding excess RBC hemoglobin to the plasma results in additional hemoglobin in the HDL fraction of both healthy volunteers and CHD patients, but still found significantly more hemoglobin in the HDL of these patients.

28A, 28B and 28C demonstrate that both non-RBC hemoglobin and haptoglobin are increased in HDL from pediatric diabetics and CHD patients. Serum was collected from 12 healthy volunteers, 14 pediatric diabetics (8 adults; 6 children), or 8 subjects with CHD or CHD equivalent disease receiving statins. Antibodies against human apoA-I were coated onto 96 well plates. Serum from these subjects was added and incubated overnight at 4 ° C. The plates were thoroughly washed and incubated overnight at 4 ° C. with goat primary antibodies against human hemoglobin or human haptoglobin. The plates were thoroughly washed and incubated for 2 hours at room temperature with a secondary antibody against goat IgG conjugated to horseradish peroxidase (HRP). The plates were thoroughly cleaned, HRP substrate was added and the optical density (OD) was measured. 28A shows non-RBC hemoglobin in μg HRP / mL. 28B shows haptoglobin in μg HRP / mL. FIG. 28C shows the result of multiplying the non-RBC hemoglobin level by the haptoglobin level. The results indicate that the values obtained for the latter method separate healthy volunteers from diabetics and CHD patients without any overlap.

FIG. 29 shows that D-4F content of D-4F in HDL-supernatant from 4 months-old apoE deficient mice (n = 8 per group) was added by adding D-4F for 2 months (+ D-4F). Proved to be significantly reduced compared to mice receiving drinking water without addition (D-4F).

FIG. 30 shows the content of myeloperoxidase in HDL-supernatant from the mice by adding D-4F for 2 months (+ D-4F) to drinking water of 4 months old apoE deficient mice (n = 8 per group). It is demonstrated that the D-4F is significantly reduced compared to mice receiving drinking water without addition (D-4F).

Detailed description of the invention

The present invention relates to a novel assay for dysfunctional high density lipoprotein (HDL). Insufficient HDL (eg, pro-inflammatory HDL) has been implicated in the pathogenesis of heart disease and in other pathological conditions characterized by inflammatory responses. Thus, assays for dysfunctional HDL may be characterized by atherosclerosis and / or other conditions characterized by an inflammatory response (eg , rheumatoid arthritis, lupus erythematosus, nodular polyarteritis, osteoporosis, atherosclerosis, stroke, Hansen's disease, tuberculosis, systemic) Lupus erythematosus, rheumatic polymyalgia, nodular polyarteritis, scleroderma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, Alzheimer's disease, chronic renal failure, diabetes, diabetic retinopathy, diabetic kidney disease, transplant rejection, graft atherosclerosis Sclerosis, reperfusion ischemia, adult respiratory syndrome, congestive heart failure, glomerulitis, metabolic syndrome, multiple sclerosis, sepsis syndrome, sickle cell disease, vascular dementia, Crohn's disease, endothelial cell dysfunction, arterial dysfunction, AIDS, rheumatoid polymorphism Myalgia, nodular polyarteritis, scleroderma, idiopathic pulmonary fibrosis, coronary artery calcification, lime Diagnosis and / or prognosis of aortic valve stenosis, osteoporosis, bacterial infections, fungal infections, autoimmune disorders, and rheumatoid arthritis, congestive heart failure, endothelial dysfunction, arterial dysfunction, viral diseases, multiple sclerosis, etc. It has a prognostic value.

There is increasing evidence suggesting that HDL-inflammatory / anti-inflammatory properties are important determinants of HDL's role in diseases such as atherosclerosis and are independent of HDL-cholesterol levels. The present invention relates to a novel assay for measuring the inflammatory properties of HDL. Assays described herein can typically be classified into two main categories:

I. Determination of HDL-associated proteins, reflecting the inflammatory properties of HDL; And

II. Determination of HDL's ability to inhibit LDL aggregation.

I. HDL  Reflecting inflammatory properties, HDL Measurement of associative proteins

A) Atherosclerosis In a mouse model of hyperlipidemia HDL  and Associated  8 proteins.

We have previously reported that the inflammatory properties of HDL are more sensitive markers for coronary artery disease (CHD) than HDL-cholesterol levels in both mice and humans. In certain embodiments, the present invention is directed, in part, to the identification of specific protein fingerprints that distinguish Group I / protective HDLs from “dysfunctional” HDLs. Although described in Examples 2 and 3 with respect to mouse HDL, the same protein / protein fingerprints and physiological mechanisms are believed to work in humans as well as other mammals. In particular, specific protein fingerprints that distinguish normal mouse HDL from mouse HDL in an atherogenic diet were identified by ProteinChip technology in conjunction with surface enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Feeding C57BL / 6J mice with an atheromatous diet for one week resulted in lower HDL-cholesterol levels, decreased paraoxonase activity, increased free radical species content and decreased ability of HDL to promote cholesterol outflow from macrophages. When the mice were converted back to normal diet for another two weeks, the pro-atherogenic properties of HDL returned to normal phenotype (see, eg, FIG. 19). We identified a total of 88 SELDI peaks with p <0.05 uniquely present in pro-inflammatory HDL from atheromatous diet fed mice compared to normal HDL from mice fed a normal diet. 74 of the 88 serum peaks returned to normal values upon dietary reversal. After further analysis to remove non-atherogenic factors and artifacts / changes resulting from short-term dietary changes, we found 24 SELDI m / z peaks that indicate proteins that are differentially associated with pro-inflammatory HDL. Was identified. Fourteen of the 24 protein peaks include three other widely used atherosclerosis / hyperlipidemia; It has been shown to be common to pro-inflammatory HDL from animal models of C57BL / 6J, LDLR deficient and apoE deficient mice on Western diets. Furthermore, an eight-protein key signature that can be used as a panel of serum biomarkers in identifying pro-inflammatory HDL in mice by protein profiling of serum samples from all four animal models (described above) One subset of SELDI m / z peaks), similar signatures can be used in humans and other mammals. These eight key signature proteins are described in detail in Example 2.

The protein signature is readily detectable (eg, using mass spectrophotometry and / or protein chip methods as described herein) and thereby can be used to identify pro-inflammatory HDL, such as in a patient. Thus, the presence or magnitude of the protein signature can be used as a diagnosis and / or prognosis for atherosclerosis and / or other conditions characterized by an inflammatory response.

In certain instances, one or more of the proteins includes (but is not limited to) electrophoresis, chromatography, and / or immunoassay which provides a rapid detection / quantification method that identifies or quantifies the strength of the protein signature in a patient. Can be detected using other standard methods.

B) Atherosclerosis In the mouse model of hyperlipidemia RBC  Hemoglobin with HDL In association  That's an amazing discovery.

As mentioned above, the inventors identified eight specific proteins that can be used individually or in combination to distinguish normal / anti-inflammatory HDL from pro-inflammatory HDL using strong anion exchange SELDI ProteinChip technology. It was. Two of the biomarker peaks (M / z 14,900 and m / z 15,600) most dramatically associated with pro-inflammatory HDL in the mouse model were further characterized. Using a micro-liquid chromatography-tandem mass spectrometer, we used SELDI peaks representing m / z 14,900 and m / z 15,600, respectively, to mouse hemoglobin alpha chain (Hb-alpha, 14.9 kDa) and mouse hemoglobin beta chain (Hb). -Beta, 15.9 kDa). Western blot analysis confirmed that Hb associates differentially with pro-inflammatory HDL compared to normal HDL. Biochemical characterization of Hb associated with HDL revealed that the Hb associated with pro-inflammatory HDL decreased pI (pI 4.0 & pI 7.0 compared to pI above 7.5 for free Hb) and the fractions found in HDL-containing fractions. It is further shown that it has unique physical and chemical properties, including association with high molecular weight complexes.

Extensive analysis of the non-RBC hemoglobin showed that the amino acid sequence was no different from RBC hemoglobin. Indeed, the altered physical properties of the non-RBC hemoglobin associated with pro-inflammatory HDL appear to be due to the tight association of hemoglobin with other proteins associated with HDL such as haptoglobin.

The surprising finding of the present invention was that there was always a small amount of non-RBC hemoglobin in plasma and serum in mice and humans. The concentration of non-RBC hemoglobin is about 10 μM. In contrast, the concentration of hemoglobin in whole blood is above 1 M. Thus, only about 0.001% of hemoglobin in total blood is outside of RBC.

It was also a surprising finding of the present invention that in normal mice and normal humans such small amounts of non-RBC hemoglobin are located in the non-lipoprotein fraction of plasma or serum. In addition, in non-atherogenic dietary mice, or in mice genetically engineered to develop atherosclerosis, in humans with diabetes or CHD, or in humans with other causes for pro-inflammatory HDL. It was also a surprising finding of the present invention that RBC hemoglobin was found in HDL.

The main form of hemoglobin found in association with HDL was oxyhemoglobin (oxyHb). Based on the pro-oxidant properties of oxyHb, our data suggest that Hb may contribute to the pro-inflammatory properties of HDL under atherogenic conditions. Furthermore, without being bound by a particular theory, the inventors believe that HDL-associated Hb can function as a novel biomarker for atherosclerosis or other pathologies characterized by an inflammatory response. Certain of these studies Details are described in Example 3.

C) Atherosclerosis In a mouse model of hyperlipidemia HDL  and Associated  Acute phase protein.

As mentioned above, we have found that hemoglobin (Hb) with physical and chemical properties distinct from erythrocytes Hb is associated with HDL in animal models of atherosclerosis and contributes to the pro-inflammatory properties of HDL under atheromatous conditions. Found that That is, for example, feeding a C57BL / 6J mouse (n = 12 per group) with an atheromatous diet for one week showed the following result compared to the C57BL / 6J mouse fed a normal diet: i) HDL- Lower cholesterol levels, ii) decreased paraoxonase activity, and iii) increased free radical species and iv) decreased ability of HDL to promote cholesterol outflow from macrophages (see, eg, FIG. 19, Example 2).

Hemopexin and haptoglobin are two well-known scavengers for Hb. Haptoglobin (Hp) is known to associate with HDL, and besides its concentration in plasma increases with hemolysis, it also increases during acute phase reactions. It was a surprising finding of the present invention that hemopexin (Hx) is also associated with pro-inflammatory HDL and similarly increases with acute phase response.

As shown in FIG. 1, Western analysis showed that the association of HDL fraction (A) with Hb, Hx, and Hp protein complexes obtained from C57BL / 6J mice fed with an atheromatous diet was normal (C). Increased compared to (> 10-fold).

Hb, Hx, and Hp protein complexes have also been shown to be associated with HDL from apoE deficient mice on normal diet. To determine if the increased levels of these HDL-associated proteins associated with the heme pathway are new effective markers for inflammation in atherosclerosis, these apoE deficient mice were apolipoprotein AI mimetic peptide D-4F for up to 21 days. (50 μg per ml of drinking water) and their HDL were analyzed for inflammatory properties and HDL-associated protein related to the heme pathway.

D-4F treatment i) converted HDL from pro-inflammatory to anti-inflammatory (eg, see FIG. 2), and ii) Hx (eg, see FIG. 3) and Hp (eg, in serum) as determined by ELISA. 4) significantly reduced levels, iii) reduced Hx and Hp protein complexes in the HDL fraction (see, eg, FIG. 5), and iv) physical and chemical properties of Hb in apoE deficient serum and apoE deficient HDL supernatant They were partially returned to normal (ie, as seen in red blood cells) (see, eg, FIG. 6). That is, in certain embodiments the present invention provides a method for detecting a heme-related protein associated with HDL which is a component of pro-inflammatory HDL.

As shown in FIG. 7, both alpha- (α-) and beta- (β-) chains of hemoglobin were increased in wild type C57BL / 6J mice fed with an atheromatous diet. In apoE deficient mice on a normal diet, the content of both the α and β chains of hemoglobin was increased and both chains were reduced by D-4F treatment.

As shown in FIG. 8, the contents of oxy-hemoglobin and met-hemoglobin in HDL were wild-type fed with normal diet for 7 days (D7C) or with atherogenic diet for 7 days (D7A) or for 15 weeks (W15A). It was measured in C57BL / 6J mice. Oxy-hemoglobin and met-hemoglobin content was also measured in apoE deficient mice (apoE) fed a normal diet.

Oxy-hemoglobin consumes nitrogen monoxide, while met-hemoglobin does not consume nitrogen monoxide. As shown in FIG. 9, the natural decomposition of chemically produced nitrogen monoxide did not change by addition of normal mouse HDL. In contrast, as shown in FIG. 10, in the decomposition of chemically produced nitrogen monoxide when oxy-hemoglobin (HbO ₂ ) was added to nitrogen monoxide (NO donor), which was chemically produced and measured as current (pA). There was a sudden and dramatic change, indicating that nitrogen monoxide was consumed by the oxy-hemoglobin. As also shown in FIG. 10, the decomposition curve did not change with the addition of phosphate buffered saline (PBS), indicating that no consumption of nitrogen monoxide occurred as a result of the addition of vehicle (PBS). In contrast, when HDL from wild-type C57BL / 6J mice fed with an atheromatous diet for 7 days (D7A HDL-Hb) showed a dramatic drop in the degradation curve, which pro-inflammatory HDL reduced nitrogen monoxide. Indicates rapid consumption.

As shown in FIG. 11, pro-atherogenic HDL from mice fed with an atheromatous diet was treated with an agent [K ₃ Fe (CN) ₆ ] which converts oxy-hemoglobin into meth-hemoglobin. The ability of pro-inflammatory HDL to rapidly consume nitric oxide was reversed.

12 shows that a normal diet was fed for 7 days (top left panel) or 15 weeks (top right panel) or an atherogenic diet was for 7 days (bottom left panel) or 15 weeks (bottom right panel). Two-dimensional gels are shown for hemoglobin and red blood cell (RBC) hemoglobin in serum from wild-type C57BL / 6J mice fed. FIG. 13 is a representation of the bottom panels of FIG. 12 fed with an atherogenic diet for 15 days (D7A) or 15 weeks of W15A in wild-type C57BL / 6J mice, except that hemoglobin staining appeared in light blue. The western blot shown in FIG. 13 was stripped off and re-probed with an antibody against haptoglobin (FIG. 14). Western blot shown in FIG. 14 was stripped off and re- probed with antibodies to hemopexin (FIG. 15).

Experiments demonstrated with FIGS. 12-15 show that hemoglobin, haptoglobin and hemopexin can be located on the same particles in atheromatous serum.

As shown in FIG. 1, these particles are mainly associated with HDL in an atheromatous diet. By supplying an atheromatous diet for 7 days to C57BL / 6J mice deficient in haptoglobin and / or hemopexin, an important role of haptoglobin in the formation of pro-inflammatory HDL was demonstrated. Briefly, normal (C) or atherogenic diets in C57BL / 6J mice that are wild type (WT) or hemopexin deficiency (Hx) or haptoglobin deficient (Hp) or both hemopexin and haptoglobin deficient (Hp / Hx). (A) was supplied for 7 days. The mice were bled and the cholesterol and heme contents were measured in the FPLC fraction of their plasma. There was virtually no associated heme in wild-type HDL. When these mice were fed an atherogenic diet for 7 days, there were associated heme in the HDL fractions (yellow bottom panel). In the absence of hemopexin, heme associated with the HDL fraction was present even in the normal diet, and the heme content in their HDL increased when these mice were fed an atheromatous diet for 7 days. In contrast, mice lacking haptoglobin did not have HDL-associated heme in the atheromatous diet (green line right bottom panel; red line right bottom panel). Hem needed haptoglobin to associate with HDL.

In another experiment, normal or atherogenicity in C57BL / 6J mice that are wild type (WT) or hemopexin deficiency (Hx) or haptoglobin deficient (Hp) or both hemopexin and haptoglobin deficient (Hp / Hx) The adult diet was fed for 7 days. The mice were bleeding and their cholesterol and free radical species (ROS) content in their FPLC fractions were measured. There was virtually no associated ROS in HDL. When these mice were fed an atheromatous diet for 7 days, there was ROS associated with the HDL fractions. In the absence of hemopexin, there was ROS associated with the HDL fractions even in the case of normal diet, and the ROS content in HDL of these mice increased when they were fed an atheromatous diet for 7 days. In contrast, mice lacking haptoglobin did not have HDL-associated ROS in atheromatous diets. That is, ROS needed haptoglobin to associate with HDL.

The data presented herein demonstrate the surprising finding that pro-inflammatory HDL can be detected by signature proteins associated with HDL or by levels of heme containing proteins associated with HDL. The present invention also predicts that the ratio of heme-containing and / or heme-binding proteins associated with HDL to the levels of these proteins in the non-lipoprotein fraction of plasma / serum is not predicted for pro-inflammatory HDL. Leads to an unexpected discovery. The present invention also leads to the unexpected finding that HDL cannot exhibit pro-inflammatory properties (such as increased ROS content) in the absence of HDL-associated heme-containing and / or heme-binding proteins.

Based on these unexpected findings, a number of assays are provided to identify pro-inflammatory (dysfunctional) HDL. Such assays include, but are not limited to, detection of one, two, three, four, five, six, seven or eight proteins associated with HDL, wherein m of the proteins the / z ratio is about: 9.3; 14.9; 15.6; 15.8; 16.2; 16.5; 18.6; 19.5, and the association of these proteins with HDL indicates that the HDL is pro-inflammatory HDL.

As mentioned above, it was a surprising finding of the present invention that proteins with m / z ratios of 14.9k and 15.6k are hemoglobin alpha and beta chains, respectively. It was also a surprising discovery that the protein having an m / z ratio of 19.5k was Group XII PLA ₂ .

In another embodiment, the assay binds to a heme-binding protein, such as a heme-containing and / or heme binding protein (such as hemoglobin, haptoglobin, hemopexin, myeloperoxidase, etc.), or soluble CD163 associated with HDL. Determining the level of a protein involves the increase of at least one, at least two, at least three, or four levels of the protein associated with the HDL (such as compared to the levels found in protective HDL). It indicates that the HDL is a pro-inflammatory HDL.

In certain embodiments, the assay further comprises adjusting heme-containing and / or heme binding proteins (eg, hemoglobin, haptoglobin, hemopexin, myeloperoxidase) levels associated with HDL to those in the non-lipoprotein fraction of HDL. Measuring compared to the level of heme-containing and / or heme-binding protein, wherein an increase in the level (eg, as compared to the level found in protective HDL) indicates that the HDL is a pro-inflammatory HDL. Indicates. In various embodiments the assays comprise the levels of heme-containing proteins associated with HDL (such as hemoglobin, haptoglobin, hemopexin, myeloperoxidase) versus those of the heme in the non-lipoprotein fraction of plasma and / or serum. Determining the ratio of the levels of the containing and / or heme-binding protein, wherein the ratio for at least one, at least two, at least three, or at least four of said proteins is about 0.2, 0.3, 0.4 A value of at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1 indicates that the HDL is pro-inflammatory HDL.

In various embodiments the predictive value of the assay is improved by measuring at least two heme-containing and / or heme-binding proteins. In various embodiments other factors may be combined and / or added to the measurement of two or more heme-containing and / or heme-binding proteins. That is, for example, descriminant function analysis or cluster analysis can be used to determine additional factors for each of the measured proteins. In one exemplary embodiment, the product of the content of two or more of heme-containing or heme-binding proteins, such as as shown in the example set forth in FIG. 28C, may be calculated (eg, the values for each Multiplication to determine the value obtained). Using the example shown in FIG. 28C, as shown in the example shown in FIG. 27, when hemolysis occurred in vitro during or after blood was drawn out of the subject, such an assay may yield a predictive value of the assay. Can be improved. That is, for example, an assay metric can be obtained by calculating the product of the hemoglobin associated with HDL x the value of haptoglobin associated with HDL (all in μg HRP / mL).

In various embodiments the assay involves measuring the level of heme associated with HDL and / or the iron content of HDL, and / or measuring the level of iron-containing protein associated with HDL, wherein the level of these measurements is ( Increased, eg, relative to the level found in protective HDL, indicates that the HDL is pro-inflammatory HDL.

In addition, assays are provided to identify / quantify the nitrogen monoxide consumption capacity of HDL.

II . HDL LDL  Determination of the ability to inhibit aggregation.

In various embodiments the invention also relates to the surprising finding that pro-inflammatory HDL does not prevent LDL aggregation, while anti-inflammatory HDL prevents LDL aggregation. That is, LDL aggregation assays provide a convenient assay and detection means for pro-inflammatory HDL. Various LDL aggregation assays are known to those skilled in the art and are not intended to limit the invention to any particular aggregation assay or format. One exemplary aggregation assay protocol is shown in Example 4.

As shown in FIG. 16, this assay enabled easy detection of pro-inflammatory HDL from subjects with CHD or CHD equivalent disease, which values were totally different from those obtained with HDL taken from healthy normal volunteers. . As shown in FIG. 17, the assay enabled easy detection of pro-inflammatory HDL from apoE deficient mice (mouse model of atherosclerosis), which values were completely different from those obtained with normal mouse HDL. Furthermore, the assay provided a simple means of validating the treatment for apoE deficient mice with apoA-I mimetic peptide D-4F.

III . remind Assays  Use.

That is, the present invention provides a number of assays for the detection and / or quantification of protective and / or dysfunctional (pro-inflammatory) HDL, which assays may be characterized by atherosclerosis or other conditions characterized by an inflammatory response (eg, , Atherosclerosis, Stroke, Hansen's disease, Tuberculosis, Systemic lupus erythematosus, Rheumatoid polymyalgia, Nodular polyarteritis, Scleroderma, Idiopathic pulmonary fibrosis, Chronic obstructive pulmonary disease, Alzheimer's disease, AIDS, Rheumatoid polymyalgia, Nodular polyarthritis, Diagnostic and / or prognostic methods for the detection of scleroderma, idiopathic pulmonary fibrosis, coronary artery calcification, calcified aortic stenosis, osteoporosis, bacterial infections, viral infections, fungal infections, autoimmune disorders, and rheumatoid arthritis, Crohn's disease, etc. . The assay is also very useful for detecting people at risk for atherosclerosis and other inflammatory conditions (eg, as described above) and for monitoring the effectiveness of various treatments and treatment protocols.

In various embodiments the assays measure heme-associated HDL-associated proteins (eg, hemoglobin, and / or haptoglobin, and / or hemopexin, and / or myeloperoxidase, etc.), the ratio of plasma / serum Determination of the relative distribution of HDL-associated proteins between (non) lipid protein fractions and HDL, determination of nitric oxide consumption of pro-inflammatory HDL, and determination of inhibition of LDL aggregation of HDL.

In this regard, it is to be noted that although hemopexin is known to be a weak acute phase reactant, it has never been previously thought to be a predictor of atherosclerosis or dysfunctional HDL. Indeed, to our knowledge, its association with HDL has never been established before. The protein has been thought to function primarily with haptoglobin to remove excess heme from the circulation. That is, it was a surprising finding that hemopexin is associated with HDL and its concentration in plasma and especially the content of hemopexin associated with HDL is a high prediction for atherosclerosis and dysfunctional HDL.

As indicated above, in certain embodiments, the present invention contemplates diagnostic and / or prognostic methods for the detection of atherosclerosis or other conditions characterized by an inflammatory response. The diagnostic methods described herein are also useful for the treatment of various subjects. If a subject (such as a patient) is diagnosed with dysfunctional HDL they may be the drug (s) and / or various statins (eg, atorvastatin (Lipitor®, Pfizer), simvastatin (Zocor) that restore or increase protective HDL levels. ®, Merck), pravastatin (Pravachol®, Bristol-Myers Squibb), fluvastatin (Lescol®, Novartis), lovastatin (Mevacor®, Merck), rosuvastatin (Crestor®, Astra Zeneca), and pitavastatin ( Sankyo et al.). Such drugs (activators) that restore or increase protective HDL levels include, but are not limited to, D4F (eg, Li et al. (2004) Circulation , 110: 1701-1705), PCT / US2001 / 26497, And / or PCT / US2001 / 26497, and / or PCT / US2004 / 026288, and / or PCT / US2005 / 028294, and / or PCT / US2003 / 09988, and / or USSN 10 / 273,386, all of which are hereby incorporated by reference. Active agents described in the above).

In certain embodiments, it will be appreciated that the assays of the present invention are typically performed in the context of a differentiated diagnosis that enables a skilled physician to identify specific condition (s) characterized by an inflammatory response in the subject. .

IV . HDL Detection / quantification of associated proteins.

In various embodiments the assays of the present invention comprise one or more proteins (eg, HDL-associated proteins including but not limited to hemoglobin, haptoglobin, hemopexin, myeloperoxidase, transferrin, soluble CD163, etc.). Detection and / or quantification. These are well known proteins that are well characterized. For example, haptoglobin (see, eg, GenBank NP — 005134) is a positive acute phase protein with relatively common isotopes that typically bind to and facilitate regulation of free hemoglobin in the blood (Vlierberghe et al. (2004) Clinica Chimica Acta., 345: 35-42.). CD163 is a monocyte-macrophage specific scavenger receptor that mediates the uptake and removal of haptoglobin-hemoglobin complexes (Aristoeli et al. (2006) Atherosclerosis 184; 342-347). Hemopexin is a plasma glycoprotein of 60 K-Da (Delanghe (2001) Clinica Chimica Acta , 312: 13-23 (see, eg GenBank NP_000604, PFAM Pfam Database for Protein Family and HMMs Accession No. PF00045). If free heme is formed in plasma during the degradation of heme containing enzymes such as hemoglobin, myoglobin, or catalase, it binds to hemopexin in a ratio of 1: 1 (Delanghe and Langlois (2001) Clinica Chimica Acta , 312: 13-23; Shipulina et al. (2000) J. Protein Chem ., 19: 239-248; Solar et al. (1989) FEBS Lett., 256: 225-229; Kuzelova et al. (1997) Biochim . Biophys . Acta , 1336: 497-501). Hemopexin is an important link between heme and iron metabolism and works with other iron transporters, haptoglobin and transferrin, to maintain iron homeostasis by the liver (Delanghe and Langlois (2001) Clinica Chimica Acta , 312: 13-23).

Methods of detecting / quantifying hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like are well known to those skilled in the art. For example, a medical haptoglobin assay is performed in a differential diagnosis of: acute rheumatic disease, biliary atresia, peptic ulcer, ulcerative colitis (increased haptoglobin) or chronic liver disease, fetal erythropoiesis Acupuncture, hematoma, hemolytic anemia, hemolytic anemia due to G6PD deficiency, idiopathic autoimmune hemolytic anemia, immune hemolytic anemia, drug-induced immune hemolytic anemia, primary liver disease, and transfusion side effects (low haptoglobin levels).

Essentially any method used to detect / quantify specific proteins can be used in the methods of the invention. Such methods include, but are not limited to, capillary electrophoresis, western blot, mass spectrometry, chromatography (eg, HPLC), immunoassay, and the like.

A) Sample Collection and Processing.

Hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like are preferably quantified in biological samples (eg whole blood, plasma, etc.) taken from a mammal, more preferably a human patient. As used herein, a biological sample is a biological sample containing one or more assay proteins described herein (eg, haptoglobin and / or hemopexin) at a concentration that may be correlated with the presence and / or level of dysfunctional HDL. A sample of tissue or body fluids. Particularly preferred biological samples include, but are not limited to, whole blood, or various blood fractions (eg, plasma, serum, etc.). In certain embodiments the biological sample comprises HDL. In certain embodiments, the biological sample comprises serum or plasma, or additional samples are provided comprising serum or plasma.

The biological sample may be pretreated or concentrated, if desired, by dilution with an appropriate buffer solution. Any of a number of standard aqueous buffer solutions using one of a variety of buffers, such as physiological pH, phosphoric acid, Tris, and the like, can be used.

As indicated above, in certain embodiments, the assay is performed using whole blood, serum, or plasma. Obtaining and storing blood and / or blood products is well known to those skilled in the art. Typically blood is obtained by venipuncture. Blood can be diluted by addition of buffers or other reagents well known to those skilled in the art and can be stored for up to 24 hours at 2-8 ° C or longer periods below -20 ° C prior to measurement. In a particularly preferred embodiment, the blood or blood product (such as serum) is stored indefinitely without preservatives at -70 ° C.

In various embodiments, as described above, the sample comprises HDL and / or HDL is isolated from the sample. Isolation methods of HDL are well known to those skilled in the art and are described in the Examples herein.

B) Immunological Binding Assay.

In a preferred embodiment, any of a number of widely accepted immunological binding assays are used to detect and / or quantify protein (s) in the biological sample (e.g., see US Pat. No. 4,366,241; 4,376,110; 4,517,288 ; 4,837,168, 6,974,704, 6,964,872, 6,887,362, 6,878,558, 6,855,562, 6,849,457, 6,835,543, 6,830,731, 6,818,456, 6,818,455, 6,770,489, 6,737,277, 6,723,524, 6,682,560,6,689,648,6,689,648, For an overview of the overall immunoassay, see also,Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993);Basic and Clinical Immunology 7th edition, Stites & Terr, eds. (1991).

Immunological binding assays (or immunoassays) typically bind specifically to the analyte (in this case hemoglobin, haptoglobin, hemopexin, myeloperoxidase, etc., and / or fragment (s) thereof). Often a "capture agent" is used to immobilize it. The capture agent is a portion that specifically binds to the analyte. In a preferred embodiment, the capture agent is in this case an antibody that specifically binds to hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like and / or fragment (s) or subtypes thereof.

In this regard, it should be noted that monoclonal and polyclonal antibodies for detecting hemoglobin, haptoglobin, hemopexin and myeloperoxidase and the like are commercially available (see, eg, anti-haptoglobin antibodies) ab8968 (sheep, polyclonal), ab4248 (chicken, polyclonal), and ab13429 (mouse, monoclonal), anti-hemopexin antibodies, ab27710 (mouse, monoclonal) and ab27711 (mouse, monoclonal, Abcam, Inc.), etc.).

Immunoassays also often use labeling substances that specifically bind to and label the binding complex formed by the capture agent and the analyte. Alternatively, the labeling substance may be a third part, such as another antibody, that specifically binds to the antibody / haptoglobin and / or antibody / hemopexin.

In certain embodiments, the labeling agent comprises an anti-haptoglobin and / or anti-hemopexin and / or anti-hemoglobin, and / or anti-myeloperoxidase antibody with a label. Alternatively, the antibody may lack a label, but this time it may be bound by a labeled third antibody specific for antibodies of the species from which the antibody is derived. In other words, an anti-haptoglobin antibody modified with a detectable moiety, such as biotin, to which a third labeled molecule, such as, for example, an enzyme-labeled streptavidin, can specifically bind.

Other proteins that can specifically bind immunoglobulin constant regions, such as Protein A or Protein G, are also available as labeling materials. These proteins are normal components of the cell walls of streptococcal bacteria. They exhibit strong non-immunogenic reactivity to immunoglobulin constant regions from various species (see, for example, below). Kronval, et al . (1973) J. Immunol . 111: 1401-1406; Akerstrom, et al . (1985) J. Immunol . , 135: 2589-2542 and the like).

Throughout this assay, incubation and / or washing steps can be performed after the combination of the respective reagents. Incubation steps may vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will vary depending on the assay mode, analyte, volume of solution, concentration, and the like. Usually, the assay will be performed at ambient temperature, but can be performed over a range of temperatures, such as 4 ° C. to 40 ° C.

1) Non-competitive test method.

In various embodiments, immunoassays that detect or quantify hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like can be competitive or noncompetitive. Noncompetitive immunoassays are assays that directly measure the amount of analyte captured (in this case hemoglobin, and / or haptoglobin, and / or hemopexin, and / or myeloperoxidase, etc.). In one preferred “sandwich” assay, for example, the capture agent (eg , anti - haptoglobin antibody and / or anti-hemoglobin antibody, and / or anti-hemopexin antibody, and / or anti-myeloperoxy Multidrug antibodies) can be directly bound to the solid substrate to which they are immobilized. These immobilized antibodies then capture hemoglobin, haptoglobin, hemopexin, and / or myeloperoxidase present in the test sample. The immobilized captured protein is then bound by a labeling substance, such as a secondary haptoglobin and / or hemopexin antibody with a label.

Alternatively, the secondary antibody may lack a label, but this time it may be bound by a labeled third antibody specific for antibodies of the species from which the secondary antibody is derived. The secondary antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.

2) competitive testing.

In competitive assays, the amount of analyte (hemoglobin, haptoglobin, hemopexin, myeloperoxidase, etc.) present in the sample is determined by the capture agent (eg anti-hemoglobin, haptoglobin, hemopexin, myeloperoxidase antibody). To determine the amount of added (exogenous) analyte (e.g., haptoglobin, hemopexin, hemoglobin, myeloperoxidase, etc.) that has been replaced (or pushed away from) the analyte present in the sample by Indirectly measured. In one competitive assay, known amounts of, in this case, hemoglobin, haptoglobin, hemopexin, myeloperoxidase, and the like are added to the sample, and then the sample is captured, in this case hemoglobin, haptoglobin Contact with an antibody that specifically binds to hemopexin, myeloperoxidase, and the like. The amount of hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like bound to the antibody is inversely proportional to the concentration of the analyte present in the sample.

In certain embodiments, the antibody is immobilized on a solid substrate. The amount of hemoglobin, haptoglobin, hemopexin, and myeloperoxidase bound to the antibody is measured by measuring the amount of hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like present in the antibody / analyte complex. Or alternatively, it can be determined by measuring the amount of hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like remaining without forming a complex. The amount of hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like can be detected by providing labeled hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like.

The hapten inhibition assay is another suitable competitive assay. In this assay, known analytes, in this case hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like are immobilized on a solid substrate. Known amounts of anti-hemoglobin, haptoglobin, hemopexin, mieloperoxidase and the like are added to the sample, and the sample is then immobilized with the immobilized hemoglobin, haptoglobin, hemopexin, myeloperoxidase Contact the back. In this case, the amount of the anti-hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like bound to the immobilized hemoglobin, haptoglobin, hemopexin, myeloperoxidase and the like is determined by the amount of the analyte present in the sample. Inversely proportional to quantity Again, the amount of immobilized antibody can be detected by detecting a fraction of the immobilized antibody or a fraction of the antibody remaining in solution.

Detection can be direct when the antibody is labeled, or can be indirect by subsequently adding a labeled moiety that specifically binds to the antibody as described above.

3) RIA Protein detection by

In certain embodiments, the hemoglobin, and / or haptoglobin, and / or hemopexin, and / or myeloperoxidase content of the sample is quantified using radioimmunoassay (RIA). Detailed protocols for radioimmunoassays can be found, for example, in Sambrook et al. (1989) Molecular Cloning -A Laboratory Manual (2nd Edition) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, et al.

4) Other test methods.

In another embodiment, Western blot (immunoblotting) analysis is used to detect and quantify the presence of analyte protein (s) in a sample. The technique generally involves separating sample proteins by gel electrophoresis based on molecular weight, transferring the separated proteins to a suitable solid support (eg, nitrocellulose filter, nylon filter, or derivatized nylon filter), And incubating the sample with antibodies that specifically bind to a target analyte (eg , hemoglobin, haptoglobin, hemopexin, myeloperoxidase, etc.). Antibodies specific for the analyte (s) of interest specifically bind to the analyte present on the solid support. These antibodies can be directly labeled or alternatively detected using labeled antibodies (eg, labeled amounts of anti-mouse antibody) that specifically bind anti-haptoglobin and / or hemopexin. May be

Other assays include, but are not limited to, liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (eg, antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (Monroe, etc. (1986) Amer Clin Prod Rev 5 : 34-41, see...).

The foregoing assays are intended to be illustrative and non-limiting. Using the teachings presented herein, other assay schemes will be apparent to those skilled in the art. It should be noted that specific protocols are also presented in this example.

C. Quantification of the Test.

Assays of the present invention are quantified according to standard methods well known to those skilled in the art. Assays of the invention are typically quantified in amounts if a difference in the levels of the assayed proteins is detectable. In certain embodiments, the change may be performed by any statistical test (e.g. , t-test, analysis of variance (ANOVA), semiparametric technique, non-parametric technique (e.g. , The Wilcoxon Mann-Whitney Test, Wilcoxon Signed Ranks Test, Sign Test, Kruskal-Wallis Test, etc.) are statistically significant changes. Preferably said statistically significant change is significant at least at 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% or 99% confidence level. . In certain embodiments, the change is at least 10% change, preferably at least 20% change, more preferably at least 50% change and most preferably at least 90% change.

V. Nitric Oxide Assay.

It was also the discovery of the present invention that while pro-inflammatory HDL consumes nitrogen monoxide (and thus produces nitrates), protective HDL substantially does not consume nitrogen monoxide. In other words, the measurement of nitrogen monoxide consumption / depletion provides a quick and easy assay for protective HDL.

Methods of detecting nitrogen monoxide consumption are well known to those skilled in the art. The methods typically involve providing nitrogen monoxide or a chemical donor thereof and detecting the consumption of the nitrogen monoxide using absorption spectroscopy or electrochemical methods.

VI . LDL  Coagulation assay.

In certain embodiments, assays are provided for non-protective HDL based on the ability of the protective HDL to prevent LDL aggregation, wherein pro-inflammatory HDL does not prevent LDL aggregation. The assay generally involves contacting an LDL (eg, isolated LDL) with the HDL and determining the amount or rate of aggregation of the LDL. Methods of measuring LDL aggregation are well known to those skilled in the art.

In one simple embodiment, the LDL is simply vortexed into a solution and spectroscopically, e.g., over time for an appropriate control (e.g., the same experiment conducted with a background solution, and / or protective HDL). The aggregation rate is measured (eg, every 10 seconds) by reading the absorbance at 680 nm (see, eg, Khoo et al. (1988) Arteriosclerosis 8: 348-58 for an exemplary aggregation assay). In certain embodiments aggregation is measured using an albumin removal column such as as described in Example 4.

VII . Test Optimization.

Assays of the present invention may be optimized for use in certain situations, for example, depending on the source and / or nature of the biological sample and / or the specific test reagent, and / or available analytical equipment. That is, for example, optimization may determine the optimal conditions for binding assays, optimal sample processing conditions (such as preferred isolation conditions), antibody conditions to maximize signal-to-noise, protocols to improve throughput, and the like. May be accompanied. In addition, the assay mode may be selected and / or optimized depending on the availability of the equipment and / or reagents.

Routine selection and optimization of assay schemes are well known to those skilled in the art.

VIII . Kit .

In certain embodiments, the present invention contemplates a kit for performing one or more of the assays described herein. Typically such kits will include one or more reagents for the detection of hemoglobin, and / or myeloperoxidase, and / or haptoglobin and / or hemopexin. Such reagents include, but are not limited to, antibodies specific for the various proteins. In certain embodiments the kits comprise one or more reagents for performing an LDL aggregation assay or a nitric oxide consumption assay.

The kits may optionally include additional materials for the collection of blood, and / or the isolation of HDL and / or LDL, and the like.

In addition, the kits may optionally include instructions for use containing instructions (ie, protocols) for practicing the method of the present invention. Preferred instructions for use provide protocols using kit contents for haptoglobin and / or hemopexin levels determination. The instructions for use typically include but are not limited to handwritten or printed materials. Any medium is contemplated in the present invention as long as it can store these instructions and deliver them to the end user. Such media include, but are not limited to, electronic storage media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD ROMs), and the like. Such media may also include the addresses of Internet sites providing such instructional materials.

The following examples are provided for illustration and are not intended to limit the claimed invention.

Example  One

hemoglobin, Haptoglobin  And Hemopexin  Measure

The ELISA protocols for haptoglobin and hemopexin are shown below, as are the protocols for HDL-supernatant preparation and apoA-I associated immunoabsorbed proteins.

Human hemoglobin ELISA

Materials for Hemoglobin ELISA. material Dealer Catalog number One 2 3 4 5 6 HRP conjugated goat anti-human hemoglobin plate reader (such as VersaMax Tunable) round bottom 96 well plate, polypropylene, non-sterile Immulon plate TMB H ₂ SO ₄ Novus Biologicals Molecular Devices Fisher Nunc Fisher Fisher ab19362 12-565-502 F25-034-03 Note: All reagents reach room temperature before use

magnetism Bead  Reagent HDL  Isolation

1. Separate plasma or serum from blood samples by centrifugation for 20 minutes at 2300 rpm at 5 ° C. using a green top tube or serum separation tube.

2. Remove the supernatant (serum or plasma). Note: If the sample is already frozen, thaw it and spin in a centrifuge for 5 minutes at 12,000 rpm at room temperature. This will precipitate any particles present in serum or plasma that may affect this assay.

3. Add supernatant (up to 250 μl / well) to a clear round bottom 96 well plate.

4. Add 1/5 of the magnetic bead reagent (up to 50 μl / well) of the total volume of the supernatant to each sample and mix. Leave for 5 minutes.

5. Place the plate on top of the magnetic particle concentrator for 5 minutes.

6. Take the supernatant and add to the microcentrifuge tube. Note: The supernatant contains HDL. ApoB containing particles were removed.

7. Remove any beads by centrifugation at 5 ° C. at 12,000 rpm for 5 minutes.

8. Remove the supernatant.

Buffer  Produce

1. Preparation of Coating Buffer: 25 mL ddH ₂ 0 + 8 mL Buffer A + 17 mL Buffer B (Buffer A: 0.2M Na ₂ CO ₃ ; Buffer B: 0.2M NaHCO ₃ )

2. Preparation of the wash buffer: 0.75 mL Tween 20 + 150 mL 10X PBS + 1350 mL ddH ₂ O.

3. Preparation of blocking / dilution buffer: 20 mL 10 × PBS + 10 mL Tween 20 + 0.5 g BSA + 170 mL ddH ₂ O.

4. Preparation of reaction stop solution: 1.38 mL H ₂ SO ₄ + 48.62 mL H ₂ O.

Preparation of Samples and Standard Curves

1. Dilute the sample (serum or HDL supernatant) 1: 200 with coating buffer (2.5 μL HDL into 497.5 μL coating buffer).

2. Make hemoglobin at a concentration of 1000 ng / mL in coating buffer (2 μl hemoglobin into 2 mL coating buffer).

3. Prepare 500, 250, 125, 62.5, 31.25, 15.63 ng / mL solutions by diluting the 1000 ng / mL solution 1: 2 step with coating buffer to prepare three standard points.

4. Include wells containing only coating buffer as blank.

Sample and Standards  Added to the plate

1. Add three standards of each standard and 110 μl of each sample to a round bottom 96 well, polypropylene plate.

2. Using a multichannel pipette, remove 100 μl from each well and add to the Immulon plate.

3. Incubate overnight at 4 ° C.

4. Whisk the antigen into the biohazard disposal container.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

Blocking and primary antibodies

1. Add 200 μl of blocking buffer to each well and incubate 1 hr at room temperature.

2. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

3. Dilute 1: 10000 HRP conjugated goat anti-human hemoglobin (1 μL antibody in 10 mL dilution buffer).

4. Add 50 μl of 1: 20000 dilution to each well.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

TMB  And reaction stopper

1. Mix 1 part TMB A and 1 part TMB B.

2. Add 100 μl of TMB mixture to each well and incubate for 20 minutes.

3. Add 100 μl of reaction stopper to each well.

Reading in Plate Reader

1. Read at 450 nm wavelength in a plate reader.

Haptoglobin ELISA

Material for Haptoglobin ELISA. material Dealer Catalog number AssayMax Human Haptoglobin ELISA Kit Plate Reader (eg VersaMax Tunable) Round Bottom 96 Well Plate, Polypropylene, Non-Sterile AssayPro Molecular Devices Fisher EH1003-1 12-565-502 Note: All reagents reach room temperature before use

magnetism Bead  Reagent HDL  Isolation

1. Separate plasma or serum from blood samples by centrifugation for 20 minutes at 2300 rpm at 5 ° C. using a green top tube or serum separator tube.

2. Remove the supernatant (serum or plasma). Note: If the sample is already frozen, thaw it and spin for 5 minutes in a centrifuge at 12,000 rpm at room temperature. This will precipitate any particles present in serum or plasma that may affect this assay.

3. Add supernatant (up to 250 μl / well) to a clear round bottom 96 well plate.

4. Add 1/5 of the magnetic bead reagent (up to 50 μl / well) of the total volume of the supernatant to each sample and mix. Leave for 5 minutes.

5. Place the plate on top of the magnetic particle concentrator for 5 minutes.

6. Take the supernatant and add to the microcentrifuge tube. Note: The supernatant contains HDL. ApoB containing particles were removed.

7. Remove any beads by centrifugation at 5 ° C. at 12,000 rpm for 5 minutes.

8. Remove the supernatant.

Of buffers  Produce

1. Dilute wash buffer concentrate (provided in the kit) 1:10 with reagent grade water.

2. Dilute EIA diluent concentrate (provided in the kit) to 1:10 with reagent grade water.

Plasma / serum or HDL Supernatant  Produce

1. Dilute 1: 4000 plasma / serum or HDL supernatant into EIA diluent (provided in the kit).

2. Start by diluting 1: 100 (5 μl HDL into 495 μl diluent).

3. Dilute 1: 100 dilution 40-fold (12.5 μl of 1: 100 dilution into 487.5 μl diluent).

Manufacture of standard curves

1. Reconstitute 100 μg of haptoglobin standard (provided in the kit) with 2 mL of EIA diluent to a concentration of 50 μg / mL.

2. Allow the standard to stand for 10 minutes with gentle stirring before making the dilution.

3. Dilute the standard solution (50 μg / mL) by 1: 4 with EIA diluent to prepare 12.5, 3.13, 0.78, 0.195, and 0.049 μg / mL solutions to prepare three reference points.

Samples added to the plate

1. Dilute biotinylated haptoglobin (provided in the kit) with 4 mL EIA diluent.

2. Add three standards of each standard and 35 μl of each sample to a round bottom 96 well, polypropylene plate.

3. Using a multichannel pipette, take 25 μL from each well and add to human haptoglobin microplates (provided in the kit).

4. Using a multichannel pipette, add 25 μl of biotinylated haptoglobin to each well.

5. Wrap wells with sealing tape (provided in the kit) and incubate 1 hr at room temperature with gentle stirring

6. Wash 5 times with 200 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

Streptavidin - Peroxidase  Addition of conjugates

1. Briefly spin down the streptavidin-peroxidase conjugate.

2. Dilute the conjugate 1: 100 with EIA diluent.

3. Add 50 [mu] l of the SP conjugate to each well and incubate for 30 minutes.

4. Wash 5 times with 200 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

Add substrate and reaction stop

1. Add 50 μl of Chromogen substrate (provided in the kit) per well and incubate for 8 minutes.

2. Add 50 μl of reaction suspension (provided in the kit) to each well.

Reading in Plate Reader

1. Read at 450 nm wavelength in a plate reader.

Hemopexin ELISA

Ingredients for hemopexin assay. material Dealer Catalog number Hemopexin plate reader from HRP conjugated chicken anti-human hemopexin human plasma (eg VersaMax Tunable) round bottom 96 well plate, polypropylene, non-sterile Immulon plate TMB H ₂ SO ₄ Immunology Consultants Laboratory Athens Research & Technology, Inc. Molecular Devices Fisher Nunc Fisher Fisher CHX-80P 12-565-502 F25-034-03 Note: All reagents reach room temperature before use

magnetism Bead  Reagent HDL  Isolation

1. Separate plasma or serum from blood samples by centrifugation for 20 minutes at 2300 rpm at 5 ° C. using a green top tube or serum separator tube.

2. Remove the supernatant (serum or plasma). Note: If the sample is already frozen, thaw it and spin for 5 minutes in a centrifuge at 12,000 rpm at room temperature. This will precipitate any particles present in serum or plasma that may affect this assay.

3. Add supernatant (up to 250 μl / well) to a clear round bottom 96 well plate.

4. Add 1/5 of the magnetic bead reagent (up to 50 μl / well) of the total volume of the supernatant to each sample and mix. Leave for 5 minutes.

5. Place the plate on top of the magnetic particle concentrator for 5 minutes.

6. Take the supernatant and add to the microcentrifuge tube. Note: The supernatant contains isolated HDL. ApoB containing particles were removed.

7. Remove any beads by centrifugation at 5 ° C. at 12,000 rpm for 5 minutes.

8. Remove the supernatant.

Of buffers  Produce

1. Preparation of Coating Buffer: 25 mL ddH ₂ 0 + 8 mL Buffer A + 17 mL Buffer B (Buffer A: 0.2M Na ₂ CO ₃ ; Buffer B: 0.2M NaHCO ₃ ).

2. Preparation of the wash buffer: 0.75 mL Tween 20 + 150 mL 10X PBS + 1350 mL ddH ₂ O.

3. Preparation of blocking / dilution buffer: 20 mL 10 × PBS + 10 mL Tween 20 + 0.5 g BSA + 170 mL ddH ₂ O.

4. Preparation of reaction stop solution: 1.38 mL H ₂ SO ₄ + 48.62 mL H ₂ O.

Preparation of Samples and Standard Curves

1. Dilute sample (plasma or HDL supernatant) 1: 200 with coating buffer (2.5 μL HDL into 497.5 μL coating buffer).

2. Make hemopexin at a concentration of 1000 ng / mL in coating buffer (2 μL hemopexin into 2 mL coating buffer).

3. Dilute 1000 ng / mL solution 1: 2 with coating buffer to prepare 500, 250, 125, 62.5, 31.25, 15.63 ng / mL solutions to prepare three reference points.

4. Include wells containing only coating buffer as blank.

Sample and Standards  Added to the plate

1. Add three standards of each standard and 110 μl of each sample to a round bottom 96 well, polypropylene plate.

2. Using a multichannel pipette, remove 100 μl from each well and add to the Immulon plate.

3. Incubate overnight at 4 ° C.

4. Whisk the antigen into the biohazard disposal container.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

Blocking and primary antibodies

1. Add 200 μl of blocking buffer to all wells and incubate for 1 hr at room temperature.

2. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

3. Dilute 1: 20000 HRP conjugated chicken anti-human hemopexin (1 μL antibody in 20 mL dilution buffer).

4. Add 50 μl of 1: 20000 dilution to each well.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

TMB  And addition of reaction stopper

1. Mix 1 part TMB A and 1 part TMB B.

2. Add 100 μl of TMB mixture to each well and incubate for 20 minutes.

3. Add 100 μl of reaction stopper to each well.

Reading in Plate Reader

1. Read at 450 nm wavelength in a plate reader.

ApoA -I associated immunosorbed protein hemoglobin ELISA

Materials for ApoA-I Associative Immunosorbed Protein Hemoglobin ELISA material Dealer Catalog number 1 2 3 4 5 6 7 8 HRP conjugated goat anti-human hemoglobin total human apolipoprotein A1 ELISA kit human hemoglobin plate reader (eg VersaMax Tunable) round bottom 96 well plate, polypropylene, non-sterile Immulon plate TMB H ₂ SO ₄ Novus Biologicals Alerchek Athens Research & Technology, Inc. Molecular Devices Fisher Nunc Fisher Fisher Ab19362 12-565-502 F25-034-03 Note: All reagents reach room temperature before use

magnetism Bead  Reagent HDL  Isolation

1. Separate plasma or serum from blood samples by centrifugation for 20 minutes at 2300 rpm at 5 ° C. using a green top tube or serum separator tube.

2. Remove the supernatant (serum or plasma). Note: If the sample is already frozen, thaw it and spin for 5 minutes in a centrifuge at 12,000 rpm at room temperature. This will precipitate any particles present in serum or plasma that may affect this assay.

3. Add supernatant (up to 250 μl / well) to a clear round bottom 96 well plate.

4. Add 1/5 of the magnetic bead reagent (up to 50 μl / well) of the total volume of the supernatant to each sample and mix. Leave for 5 minutes.

5. Place the plate on top of the magnetic particle concentrator for 5 minutes.

6. Take the supernatant and add to the microcentrifuge tube. Note: The supernatant contains HDL. ApoB containing particles were removed.

7. Remove any beads by centrifugation at 5 ° C. at 12,000 rpm for 5 minutes.

8. Remove the supernatant.

Of buffers  Produce

1. Preparation of Coating Buffer: 25 mL ddH ₂ O + 8 mL Buffer A + 17 mL Buffer B (Buffer A: 0.2M Na ₂ CO ₃ ; Buffer B: 0.2M NaHCO ₃ ).

2. Preparation of the wash buffer: 0.75 mL Tween 20 + 150 mL 10X PBS + 1350 mL ddH ₂ O.

3. Preparation of blocking / dilution buffer: 20 mL 10 × PBS + 10 mL Tween 20 + 0.5 g BSA + 170 mL ddH ₂ O.

4. Preparation of reaction stop solution: 1.38 mL H ₂ SO ₄ + 48.62 mL H ₂ O

Preparation of Samples and Standard Curves

1. Dilute the sample (serum or HDL supernatant) 1: 200 with coating buffer (2.5 μL HDL into 497.5 μL coating buffer).

2. Make hemoglobin at a concentration of 1000 ng / mL in coating buffer (2 μl hemoglobin into 2 mL coating buffer).

3. Dilute 1000 ng / mL solution 1: 2 with coating buffer to prepare 500, 250, 125, 62.5, 31.25, 15.63 ng / mL solutions to prepare three reference points.

4. Include wells containing only coating buffer as blank.

Sample and Standards  Added to the plate

1. Add three standards of each standard and 110 μl of each sample to a round bottom 96 well, polypropylene plate.

2. Using a multichannel pipette, remove 100 μL from each well and add it to an anti-human ApoA1 coated microwell plate (provided in the A1 kit).

3. Incubate overnight at 4 ° C.

4. Whisk the antigen into the biohazard disposal container.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

Blocking and primary antibodies

1. Add 200 μl of blocking buffer to all wells and incubate for 1 hr at room temperature.

2. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

3. Dilute 1: 10000 HRP conjugated goat anti-human hemoglobin (1 μL antibody in 10 mL dilution buffer).

4. Add 50 μl of 1: 20000 dilution to each well.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

TMB  And reaction stopper

1. Mix 1 part TMB A and 1 part TMB B.

2. Add 100 μl of TMB mixture to each well and incubate for 20 minutes.

3. Add 100 μl of reaction stopper to each well.

Reading in Plate Reader

1. Read at 450 nm wavelength in a plate reader.

ApoA -I associated immunosorbed protein Haptoglobin ELISA

Material for ApoA-I associated immunosorbed protein haptoglobin ELISA. material Dealer Catalog number HRP conjugated anti-human haptoglobin total human apolipoprotein A1 ELISA kit plate reader (eg VersaMax Tunable) round bottom 96 well plate, polypropylene, non-sterile Biogenesis Alerchek Molecular Devices Fisher    12-565-502 Note: All reagents reach room temperature before use

magnetism Bead  Reagent HDL  Isolation

1. Separate plasma or serum from blood samples by centrifugation for 20 minutes at 2300 rpm at 5 ° C. using a green top tube or serum separator tube.

2. Remove the supernatant (serum or plasma). Note: If the sample is already frozen, thaw it and spin for 5 minutes in a centrifuge at 12,000 rpm at room temperature. This will precipitate any particles present in serum or plasma that may affect this assay.

3. Add supernatant (up to 250 μl / well) to a clear round bottom 96 well plate.

4. Add 1/5 of the magnetic bead reagent (up to 50 μl / well) of the total volume of the supernatant to each sample and mix. Leave for 5 minutes.

5. Place the plate on top of the magnetic particle concentrator for 5 minutes.

6. Take the supernatant and add to the microcentrifuge tube. Note: The supernatant contains isolated HDL. ApoB containing particles were removed.

7. Remove any beads by centrifugation at 5 ° C. at 12,000 rpm for 5 minutes.

8. Remove the supernatant.

Of buffers  Produce

Preparation of Coating Buffer: 25 mL ddH ₂ O + 8 mL Buffer A + 17 mL Buffer B

(Buffer A: 0.2M Na ₂ CO ₃ ; Buffer B: 0.2M NaHCO ₃ ).

2. Preparation of the wash buffer: 0.75 mL Tween 20 + 150 mL 10X PBS + 1350 mL ddH ₂ O.

3. Preparation of blocking / dilution buffer: 20 mL 10 × PBS + 10 mL Tween 20 + 0.5 g BSA + 170 mL ddH ₂ O.

4. Preparation of reaction stop solution: 1.38 mL H ₂ SO ₄ + 48.62 mL H ₂ O.

HDL Supernatant  Produce

1. Dilute HDL supernatant 1: 4000 with coating buffer.

2. Start at 1: 100 dilution (5 μL HDL into 495 μL buffer).

3. Dilute 1: 100 dilution 40-fold (12.5 μl of 1: 100 dilution into 487.5 μl buffer).

Preparation of standard curves.

1. Reconstitute 100 μg of haptoglobin standard (provided in the kit) with 2 mL of buffer to a concentration of 50 μg / mL.

2. Allow the standard to stand for 10 minutes with gentle stirring before making the dilution.

3. Dilute the standard solution (50 μg / mL) 1: 4 with buffer to prepare 12.5, 3.13, 0.78, 0.195, and 0.049 μg / mL solutions to prepare three reference points.

Sample and Standards  Added to the plate

1. Add three standards of each standard and 110 μl of each sample to a round bottom 96 well, polypropylene plate.

2. Using a multichannel pipette, remove 100 μL from each well and add it to an anti-human ApoA1 coated microwell plate (provided in the A1 kit).

3. Incubate overnight at 4 ° C.

4. Whisk the antigen into the biohazard disposal container.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

Blocking and primary antibodies

1. Add 200 μl of blocking buffer to all wells and incubate for 1 hr at room temperature.

2. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

3. Dilute HRP conjugated anti-human haptoglobin 1: 20000 (1 μL antibody in 20 mL dilution buffer).

4. Add 50 μl of 1: 20000 dilution to each well.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

TMB  And addition of reaction stopper

1. Mix 1 part TMB A and 1 part TMB B.

2. Add 100 μl of TMB mixture to each well and incubate for 20 minutes.

3. Add 100 μl of reaction stopper to each well.

Reading in Plate Reader

1. Read at 450 nm wavelength in a plate reader.

ApoA -I associated immunosorbed protein Hemopexin ELISA

Materials for ApoA-I Association Immunsorbed Protein Hemopexin ELISA material Dealer Catalog number HRP conjugated chicken anti-human hemopexin total human apolipoprotein A1 ELISA kit hemopexin plate reader from human plasma (eg VersaMax Tunable) round bottom 96 well plate, polypropylene, non-sterile Immulon plate TMB H ₂ SO ₄ Immunology Consultants Laboratory Alerchek Athens Research & Technology, Inc. Molecular Devices Fisher Nunc Fisher Fisher CHX-80P 12-565-502 F25-034-03 Note: All reagents reach room temperature before use

magnetism Bead  Reagent HDL  Isolation

1. Separate plasma or serum from blood samples by centrifugation for 20 minutes at 2300 rpm at 5 ° C. using a green top tube or serum separator tube.

2. Remove the supernatant (serum or plasma). Note: If the sample is already frozen, thaw it and spin for 5 minutes in a centrifuge at 12,000 rpm at room temperature. This will precipitate any particles present in serum or plasma that may affect this assay.

3. Add supernatant (up to 250 μl / well) to a clear round bottom 96 well plate.

4. Add 1/5 of the magnetic bead reagent (up to 50 μl / well) of the total volume of the supernatant to each sample and mix. Leave for 5 minutes.

5. Place the plate on top of the magnetic particle concentrator for 5 minutes.

6. Take the supernatant and add to the microcentrifuge tube. Note: The supernatant contains isolated HDL. ApoB containing particles were removed.

7. Remove any beads by centrifugation at 5 ° C. at 12,000 rpm for 5 minutes.

8. Remove the supernatant.

Of buffers  Produce

Preparation of Coating Buffer: 25 mL ddH ₂ O + 8 mL Buffer A + 17 mL Buffer B

(Buffer A: 0.2M Na ₂ CO ₃ ; Buffer B: 0.2M NaHCO ₃ ).

2. Preparation of the wash buffer: 0.75 mL Tween 20 + 150 mL 10X PBS + 1350 mL ddH ₂ O.

3. Preparation of blocking / dilution buffer: 20 mL 10 × PBS + 10 mL Tween 20 + 0.5 g BSA + 170 mL ddH ₂ O.

4. Preparation of reaction stop solution: 1.38 mL H ₂ SO ₄ + 48.62 mL H ₂ O.

Preparation of Samples and Standard Curves

1. Dilute the sample (HDL supernatant) 1: 200 with coating buffer (2.5 μL HDL into 497.5 μL coating buffer).

2. Make hemopexin at a concentration of 1000 ng / mL in coating buffer (2 μL hemopexin into 2 mL coating buffer).

3. Dilute 1000 ng / mL solution 1: 2 with coating buffer to prepare 500, 250, 125, 62.5, 31.25, 15.63 ng / mL solutions to prepare three reference points.

4. Include wells containing only coating buffer as blank.

Sample and Standards  Added to the plate

1. Add three standards of each standard and 110 μl of each sample to a round bottom 96 well, polypropylene plate.

2. Using a multichannel pipette, remove 100 μL from each well and add it to an anti-human ApoA1 coated microwell plate (provided in the A1 kit).

3. Incubate overnight at 4 ° C.

4. Whisk the antigen into the biohazard disposal container.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

Blocking and primary antibodies

1. Add 200 μl of blocking buffer to all wells and incubate for 1 hr at room temperature.

2. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

3. Dilute 1: 20000 HRP conjugated chicken anti-human hemopexin (1 μL antibody in 20 mL dilution buffer).

4. Add 50 μl of 1: 20000 dilution to each well.

5. Wash three times with 300 μl of wash buffer. Turn the plate over and drain the contents, tapping it 4-5 times on an absorbent paper towel to completely remove the liquid at each step.

TMB  And reaction stopper

1. Mix 1 part TMB A and 1 part TMB B.

2. Add 100 μl of TMB mixture to each well and incubate for 20 minutes.

3. Add 100 μl of reaction stopper to each well.

Reading in Plate Reader

1. Read at 450 nm wavelength in a plate reader.

It should be noted that the protocols described herein are intended to be illustrative and non-limiting. Using the teachings presented herein, other assays and assay schemes will be readily available to those skilled in the art.

result.

Plasma was collected from 10 healthy volunteers and 10 patients with proven CHD. HDL was isolated, run on native PAGE gels and immunoblotted for hemoglobin using Western analysis. As shown in the left panel of FIG. 27, dramatically more hemoglobin was found in HDL from 10 healthy volunteers compared to 10 patients with CHD. As shown in the right panel of FIG. 27, adding RBC lysate to the plasma prior to HDL isolation increased the amount of hemoglobin associated with HDL in both groups, but healthy volunteers and CHD patients under these extreme hemolytic conditions. Significant differences remained clear between the livers.

In other experiments, serum from 12 healthy volunteers and another group of 14 pediatric diabetics (8 adults and 6 children), and 8 subjects with equivalent disease according to CHD or NCEP ATP III criteria Collected them. The latter patients all received statins. Antibodies against human apoA-I were coated onto 96 well plates. Serum from these subjects was added and incubated overnight at 4 ° C. The plates were thoroughly washed and incubated overnight at 4 ° C. with primary goat antibodies against human hemoglobin or human haptoglobin. The plates were washed thoroughly and incubated for 2 hours at room temperature with secondary antibodies against goat IgG conjugated to horseradish peroxidase (HRP). The plates were thoroughly cleaned, HRP substrate was added and the optical density (OD) was measured. Non-RBC hemoglobin (μg HRP / mL) is shown in FIG. 28A. 28B shows haptoglobin (μg HRP / mL). In Fig. 28C, the product of the non-RBC hemoglobin level and the haptoglobin level is shown. The result indicates that healthy volunteers were separated from the diabetics and CHD patients without any overlap in the values obtained by the latter method.

These results indicate that the novel assays reported herein are useful in detecting subjects with diseases such as diabetes and CHD and provide a means of distinguishing them from normal individuals.

Example  2

Bulb-inflammatory In HDL  Protein profiles

We have reported that the inflammatory properties of HDL are more sensitive markers for atherosclerosis than HDL-cholesterol levels in both mice and humans. In this example, we use a ProteinChip technology coupled with surface enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to distinguish normal mouse HDL from mouse HDL on atheromatous diet. Describe fingerprints. Feeding C57BL / 6J mice with atherogenic diets for a week resulted in decreased HDL-cholesterol levels, decreased paraoxonase activity, increased free radical species and decreased ability of HDL to promote cholesterol outflow from macrophages. . When the mice were switched back to normal diet for an additional two weeks, the atheromatous promoting properties of HDL returned to normal phenotype. We identified a total of 88 SELDI peaks with p <0.05 differentially present in pro-inflammatory HDL from atheromatous diets compared to normal HDL from mice fed a normal diet. 74 of the 88 serum peaks returned to normal levels upon dietary conversion. After further analysis to remove artifacts / changes resulting from short-term dietary changes and non-atherogenic factors, we found 24 SELDI m / z peaks showing proteins associated differently with pro-inflammatory HDL. I identified them. Fourteen of the 24 protein peaks have been shown to be common to pro-inflammatory HDL from three different widely used animal models: atherosclerosis / hyperlipidemia; C57BL / 6J, LDLR deficient and apoE deficient mice on Western diet. Furthermore, by protein profiling of serum samples from all four animal models, an 8-protein core signature that can be used as a panel of serum biomarkers to identify pro-inflammatory HDL (14 SELDI m / s described above) subset of z peaks).

Experimental procedure

Animal experiment.

C57BL / 6J, LDLR deficient / C57BL / 6J and apoE deficient / C57BL / 6J female mice, 8-12 weeks old, were used in this experiment. Mice were fed one of three diets during the period described above: normal diet (Ralston Purina Mouse Chow), or 15.8% fat, 1.25% cholesterol, and 0.5% cholic acid (w / w / w) ), Or a Western diet (Teklad / Harlan, Madison WI, diet No. 88137; 42% fat, 0.15% cholesterol, w / w). Serum samples were isolated from overnight fasted mice, cryopreserved in 10% sucrose as previously described (Navab et al. (2000) J. Lipid Res . , 41: 1481-1494), and stored at -80 ° C until use.

Lipoprotein  Isolation.

Serum samples were fractionated with a gel permeation high performance liquid chromatography (FPLC) system consisting of a series of dual Pharmacia Superose 6 columns. Serum (0.5 ml) was pumped at a flow rate of 0.5 ml / min with a non-metallic Beckman HPLC pump, eluted with sterile PBS and fractionated every 1 ml. For each fraction, cholesterol content was analyzed using cholesterol reagents (Thermo, Louisville, CO) and protein content using BCA assay (Promega, Madison, WI) according to the manufacturer's protocol.

SELDI  Analysis and In vitro  For assay HDL  Isolation.

HDL was newly isolated using LipiDirect HDL reagent (Polymedco, Cortland Manor, NY) following the manufacturer's protocol. Cholesterol content analysis and BCA protein assays were performed on the supernatants containing HDL and used within 48 hours after isolation.

HDL  Active in Oxygen species  ( ROS ).

As previously described (Navab et al. (2001) J. Lipid Res . , 42: 1308-1317), 2,7,7'dichlorofluorescein diacetate (H ₂ DCFDA: Invitrogen, Carlsbad, Calif.) To determine the ROS content in HDL. Briefly, HDL was incubated at 37 ° C. for 30 minutes with H ₂ DCFDA (10 μg / ml) in methanol. Fluorescence intensity was measured at 485 nm / 525 nm to detect DCF formation as a labeling factor of ROS.

Paraoxonase  ( PON ) black.

PON activity in HDL was measured as previously described (Van Lenten et al . (1995) J. Clin . Invest . , 96: 2758-2767). HDL was incubated with paraoxon and PON activity was analyzed by measuring the increase in absorbance at 405 nm over 12 minutes. The unit of PON activity was defined as the formation of 1 nmol of 4-nitrophenol per minute per ml of added HDL.

Cholesterol spills.

Cell cholesterol efflux was performed as previously described (Navab et al. (2004) Circulation , 109: 3215-3220). Briefly, mouse RAW264.7 cells were cultured in 24-well tissue culture plates and grown overnight in DMEM medium containing 10% FBS. Cells were washed with serum free medium and ³ H-cholesterol (0.5 μCi / ml) and acetylated LDL (50 μg / ml) in medium containing 0.5% fatty acid depleted BSA (Sigma, St. Louis, MO) I stayed together overnight. Labeled cells were washed, resuspended in medium containing 0.5% BSA and incubated with HDL at 37 ° C. for 6 hours. Cholesterol efflux is expressed as a percentage of the total radioactivity released into the medium.

SELDI  Sample preparation for analysis.

Serum and HDL samples were processed on strong anion-exchange (Q10) chips according to the manufacturer's protocol (Ciphergen Biosystems, Fremont, CA). Briefly, the Q10 array spots were equilibrated with binding buffer (1 × PBS / 0.1% Triton X-100, pH 7) for 15 minutes at room temperature in a humidification chamber. Each sample was first diluted 1: 5 with 9 M urea / 2% Chaps / 50 mM Tris.HCl, pH 9.0 and further diluted 1:25 with binding buffer. Five microliters of each diluted sample were spotted onto the equilibrated Q10 protein array chip and incubated for 30 minutes at room temperature in a humidity chamber. The chip was washed twice with binding buffer and once with HPLC H ₂ O and then air-dried. The chip was treated with a sinapinic acid solution, which was sequentially treated with 0.5 μl of 100% saturated solution and then 1 μl of 50% saturated solution. The cinafinic acid solution is freshly prepared by saturating the EAM solution (50% acetonitrile and 0.5% trifluoroacetic acid) with cinafinic acid (3,5-dimethoxy-4-hydroxycinnamic acid).

Ciphergen Proteinchip SELDI - TOF - MS  analysis.

The array was analyzed by Ciphergen ProteinChip Reader (Model PB SII). An average of 65 laser shots were used at laser intensities of 230-280 arbitrary units to generate mass spectra of proteins. For data acquisition of low molecular weight proteins, the detection size range was set to 2-18 kDa and the maximum size was 25 kDa. For high molecular weight proteins, the detection size range was set to 20 to 150 kDa and the maximum size was 250 kDa. The mass-to-charge ratio (m / z) of each protein captured on the array surface was measured according to externally calibrated standards (Ciphergen Biosystems): bovine insulin (5,733.6 Da), human ubiquitin (8,564.8 Da), bovine Cytochrome c (12,230.9 Da), bovine superoxide dismutase (15,591.4 Da), bovine-lactoglobulin A (18,363.3 Da), horseradish peroxidase (43,240 Da), BSA (66,410 Da), and chicken cornalbumin ( 77,490 Da).

Statistical analysis.

The data were analyzed using ProteinChip data analysis software version 3.2 (Ciphergen Biosystems). For each comparison, the original intensity data was normalized using the total ion current of all profiles in the groups. Peak intensities were normalized to a total ion current of m / z at 3,000 to 25,000 Da for the low molecular weight range and 4,000 to 250,000 Da for the high molecular weight range. The Biomarker Wizard application (Nonparametric Calculation; Ciphergen Biosystems) was used to compile all spectra and autodetect the quantified mass peaks. Sample statistical treatments were performed on the profile groups (anti-inflammatory HDL versus pro-inflammatory HDL; normal serum versus atheromatous serum). Protein differences (fold changes) were calculated within the various groups. If a statistically significant difference in intensity was observed (p <0.05) compared to one group, the protein was considered to be associated differentially between the two groups.

result

Model system.

Hedrick et al. Reported that short-term feeding (less than 7 days) of atherogenic diets in C57BL / 6J low density lipoprotein receptor-deficient (LDLR deficient) mice resulted in a dramatic decrease in plasma PON activity and amount and an increase in plasma and HDL lipid hydroperoxide. Induced (Hedrick et al. (2000) Arterioscler . Thromb . Vasc . Biol . , 20: 1946-1952). Interestingly, when the mice ingested the atherogenic diet for 7 days and then switched to the normal diet for 3 days, the PON amount and activity returned to normal levels. However, Hedrick et al. Noted that a three-day transition to normal diet did not result in complete recovery of all HDL properties (same reference above). In an effort to identify differentially associated proteins in pro-inflammatory HDL, we used a similar mouse model system. C57BL / 6J mice fed a normal diet had anti-inflammatory HDL, while C57BL / 6J mice fed an atheromatous diet had pro-inflammatory HDL (Shih et al . (1996) J. Clin . Invest . , 97: 1630-1639). As shown in Figures 18 and 19, changing the diet from an atherogenic diet to a normal diet for another 14 days on day 7 results in HDL cholesterol (Figures 18 and 19, lower right), reactive oxygen species content (Figure 19, upper left). ), PON activity (FIG. 19, top right), and HDL-mediated cholesterol efflux (FIG. 19, bottom left) returned to normal levels. We describe these three experimental conditions (normal diets for the entire experimental period that was 21 days or less in some experiments, atheromatous diets for the entire experimental period that were 21 days or less in some experiments, or 7 days of atheromatous diets). And then using an additional 14 days of normal diet) would provide a system to identify proteins that associate with or separate from HDL when HDL is converted to a pro-inflammatory state.

Specific SELDI  Peaks are pro-inflammatory HDL  Are discriminated against.

We first used SELDI analysis to identify proteins that are differentially associated with HDL in atheromatous diets. Eight-week-old female C57BL / 6J mice (n = 8 per group) added after 7-day normal diet (C), 7-day atheromatous diet (A), 21-day normal diet (CC), or 7-day atheromatous diet One of the normal diets (AC) was fed for 14 days. Serum samples from each dietary group (obtained after overnight fasting) were collected at the end of each period. HDL from each serum was isolated using LipiDirect HDL reagent and subjected to SELDI analysis to atheromatous serum and HDL from mice on atheromatous diet compared to serum and HDL from mice receiving a normal diet. Protein profiles in were identified. Protein profiles from individual serum samples (n = 8) from each group were obtained. Protein profiles in the 'C' group were compared with protein profiles in the 'A' group, and protein profiles in the 'CC' group were compared with protein profiles in the 'AC' group. In the first set of assays, the ciphergens, as previously described, differentiated detected m / z peaks in HDL from atheromatous serum ('A' group) compared to HDL from normal serum ('C' groups). Further statistical analysis was performed with ProteinChip software (Kozak et al. (2003) Proc . Natl . Acad . Sci ., USA , 100: 12343-12348). A total of 88 peaks were detected in HDL from mice given a 21 day atheromatous diet (p <0.05) significantly different from HDL from mice given a normal diet for 21 days.

In HDL  Protein peak is Dietary  Reflect change.

When these protein profiles were analyzed for the 'AC' group, 74 of the 88 peaks returned back to the normal levels seen in HDL on normal diet, indicating that these peaks were anti-inflammatory to pro-inflammatory of HDL. And protein profiles associated with the reverse conversion (Table 12).

Short- and long-term dietary-induced progenitors-inflammatory HDL  In all there are common protein profiles.

Also, HDL in atheromatous sera from C57BL / 6J mice (n = 8) fed with atheromatous diet (W15A) for 15 weeks was serum from mice fed with normal diet (W15C) for 15 weeks. SELDI profiles were generated as compared to HDL in. We further determined peaks common in both short-term diet-induced HDL (74 peaks described above) and long-term diet-induced (15-week) HDL. We deduced that the comparison would eliminate artifacts / changes resulting from short-term dietary changes. We specifically and significantly differ in HDL from mice given an atheromatous diet for any period of time (7-day atheromatous and 15-week atheromatous diets) as compared to HDL from mice given a normal diet. A total of 24 SELDI peaks were identified (Table 7).

Since the atheromatous diet contains 0.5% cholic acid, we described all of the above described for HDL from serum samples obtained from mice fed Western diet (WD) containing no cholic acid (WD). The experiments were repeated and 21 m / z showing proteins differentially associated with HDL on both high-fat high-cholesterol diets (both atheromatous diets containing cholic acid and Western diets without kolic acid). Protein signature consisting of peaks was identified (Table 8). Compared to HDL from mice fed a normal diet, 13 protein peaks (numbers in black) strongly associated with / by: HDL from mice fed a western diet, and atheromatization There were eight isolated / reduced proteins (in parentheses) in HDL from mice fed a sex or western diet (Table 8).

In a Hyperlipidemic Mouse Model HDL  Apart from Associated  Common Protein Profiles.

To validate the identified protein profiles that distinguish HDL from a mouse model of atherosclerosis from HDL from control mice, we included a Western diet fed LDLR deficient mouse and a normal diet fed apoE deficient mouse. Other well known mouse models of atherosclerosis were used further. These mice are susceptible to atherosclerosis and have pro-inflammatory HDL (Navab et al. (2005) Ann . Med . , 37: 173-178; Shih et al . (1996) J. Clin . Invest . , 97: 1630-). 1639; Ridker (2002) Circulation , 105: 2-4). HDL samples from these atheromatous mice were subjected to SELDI analysis and the profiles were cross-validated using the C57BL / 6J mouse model. This study not only eliminated artifacts / changes caused by non-atherogenic factors and short-term dietary changes, but also identified a common biomarker for HDL in multiple atheromatous models. Fourteen of the 21 peaks associated with pro-inflammatory HDL identified in the C57BL / 6J mouse model were common to those detected in HDL from LDLR deficient and apoE deficient mice (Table 9).

Bulb-inflammatory HDL  and Associated  Potential serum Biomarkers .

To further identify serum protein profiles representing proteins associated with HDL in an atheromatous / hyperlipidemic diet, serum samples from all four mouse atherosclerosis models were subjected to SELDI analysis in the same manner as performed for HDL. Applied. 13 peaks were identified as a common biomarker in atheromatous / hyperlipidemic mouse serum by SELDI analysis (Table 10). Furthermore, eight of the 13 protein peaks detected in atheromatous / hyperlipidemic sera were common with those identified in HDL in the atheromatous / hyperlipidemic diet (14 peaks above) (Table 11). This implies that the eight proteins identified above are potential biomarkers associated with pro-inflammatory HDL detectable directly in mouse serum.

discussion

In recent years, in the fight against atherosclerosis, it has become apparent that HDL function may be a more selective therapeutic target than HDL-C (Castellani et al . (1997) J. Clin . Invest . , 100: 464-474; Navab et al . (2005) Ann . Med . , 37: 173-178; Ridker (2002) Circulation , 105: 2-4; Ansell et al. (2003) Circulation , 108: 2751-2756). Although a number of protein and enzyme activities are associated with HDL, little is known about which particular protein profile helps to better distinguish normal / anti-inflammatory HDL from pro-inflammatory HDL. We used ProteinChip technology in an established mouse model of atherosclerosis to identify m / z peaks that indicate proteins that are differentially associated with HDL in mice on atheromatous diets.

All of our experiments were performed on individual mouse samples (n = 8 in each group) and each sample was measured in triplicate. Furthermore, only the (p <0.05) m / z peaks that were significant between groups were accepted as candidate peaks for further analysis. In the first set of experiments, a total of 74 m / z peaks in the HDL (Table 12) returned to normal levels when the diet was changed from an atheromatous diet (day 7) to a normal diet for an additional 14 days. These peaks were further compared to protein profiles obtained from HDL samples from mice fed with an atheromatous diet for 15 weeks to remove any artifacts due to short term dietary changes. Interestingly, only 24 of the original 74 peaks (Table 7) were seen in HDL obtained from mice fed with an atherogenic diet for 15 weeks, with the remaining 59 m / z peaks being short-term dietary changes and / or Or may be due to non-atherogenic factors. However, it is also possible that the remaining 59 m / z peaks still show proteins associated with pro-inflammatory HDL in the early stages of atheromatous modification of HDL.

Interestingly, the three proteins m / z 5100, m / z 35300, and m / z 197000, compared to protein profiles obtained from mice fed a Western diet, were fed with an atheromatous diet (short-term Long term) did not appear in HDL from C57BL / 6J mice (Table 8). Since the main difference between the two diets used is cholates, it is possible that these three proteins exhibit proteins specific for cholate metabolism and toxicity. Identification and characterization of these proteins may prove useful for understanding HDL inflammatory properties related to bile acid metabolism. Surprisingly, protein profiles were prepared from pro-inflammatory HDL obtained from two different animal models of atherosclerosis (LDLR deficient mice on Western diet and apoE deficient mice on normal diet) and pro-inflammatory HDL from C57BL / 6J mice. Only 14 of the 21 peaks were common in these three models when compared to a common protein profile for (Table 8 and Table 9).

Serum protein profiling provides an easier and more efficient strategy for the use of biomarkers in disease diagnosis and / or drug efficacy. We identified a number of different proteins in the serum (Table 13) compared to the profiles obtained from HDL. Key sequences of 13 peaks from mouse models of all different atherosclerosis / hyperlipidemia were identified (Table 10). When we compared the final protein profile sets obtained from serum profiling and HDL profiling, we identified a set of common 8 protein peaks (Table 10). Proteins representing these eight peaks are a set of markers that are very important not only for the direct analysis of serum samples for the identification of pro-inflammatory HDL, but also for further understanding of the properties of pro-inflammatory HDL. Can be formed.

Proteins showing these m / z peaks (Table 11) are differentially associated with pro-inflammatory HDL from the C57BL / 6J mouse model of hyperlipidemia and atherosclerosis. As a first screening, normal and atheromatous serum was fractionated into ion exchange columns with different pH buffers, followed by SELDI analysis to obtain the pI value of each protein peak of interest (Kozak et al. (2005) Proteomics , 5 4589-4596). Using the pi and mass information of each protein peak, we searched databases (TagIdent) to obtain candidate proteins representing the SELDI peak of interest. We concluded that hemoglobin was associated with HDL in mice fed with an atheromatous diet. Details of these studies are presented in Example 3.

In conclusion, we characterized 8 m / z SELDI biomarker peaks in pro-inflammatory HDL to confirm their differential expression in atheromatous / hyperlipidemic serum. Together, these markers will help to determine the molecular mechanisms involved in the conversion of normal / anti-inflammatory HDL to pro-inflammatory HDL in mice. Identification of markers, as well as markers in human pro-inflammatory HDL, facilitates the development of clinical assays that improve early detection of atherosclerosis and other pathologies characterized by inflammatory responses.

Example  3

Atheromatous formation Of Hyperlipidemia Dietary  High density in serum from fed mice Geology Protein and Associated  hemoglobin

In Example 2, we identified eight specific protein fingerprints that distinguish normal / anti-inflammatory HDL from pro-inflammatory HDL in a mouse model of atherosclerosis using strong anion exchange SELDI ProteinChip technology. Using a micro-liquid chromatography-tandem mass spectrometer, we used m / z 14,900 and m /, which were mouse hemoglobin alpha chain (Hb-alpha, 14.9 kDa) and mouse hemoglobin beta chain (Hb-beta, 15.9 kDa), respectively. SELDI peaks representing z 15,600 were identified. Western blot analysis confirmed the differential association of Hb with pro-inflammatory HDL compared to normal HDL. Biochemical characterization of Hb associated with HDL revealed that Hb associated with pro-inflammatory HDL was found in fractions containing reduced pI (pI 4.0 & pI 7.0 compared to pI above 7.5 in free Hb) and HDL. It is further shown that it has unique physical and chemical properties, including association with high molecular weight complexes. The main form of hemoglobin found in association with HDL was oxyhemoglobin (oxyHb). Based on the oxidation promoting properties of oxyHb, our data suggest that Hb may contribute to the pro-inflammatory properties of HDL under atherogenic conditions. Furthermore, we infer that HDL-associated Hb may function as a novel biomarker for atherosclerosis and other pathologies characterized by inflammatory responses.

Experimental procedure

Animal experiment.

C57BL / 6J female mice (n = 8 per group), 8-8 weeks of age wild type, low density lipoprotein receptor-deficient (LDLR deficiency) and apoE deficiency, were used in the experiment comparing the three mouse models. Mice in these experiments were fed one of two diets: a normal diet (Ralston Purina Mouse Chow), or an atheromatous diet containing 15.8% fat, 1.25% cholesterol, and 0.5% cholic acid w / w / w ( Teklad / Harlan Catalog). For short-term studies, mice (n = 8 per group) were fed with the diet described above for 7 days, and for long-term studies, mice were fed with the diet described above for 15 weeks. Serum samples were isolated from overnight fasted mice and cryopreserved in 10% sucrose and maintained at −70 ° C. until use.

Lipoprotein  Isolation.

Intravitreal serum samples were fractionated with a system consisting of two consecutive Pharmacia Superose 6 columns. Serum (0.5 ml) was pumped at a flow rate of 0.5 ml / min with a non-metallic Beckman HPLC pump, eluted with sterile PBS and fractionated every 1 ml. The first 10 1 mL fractions were discarded and cholesterol content analysis (Thermo, Louisville, CO) and BCA protein assay (Promega, Madison, WI) were performed according to the manufacturer's protocol for each 1 mL fraction collected thereafter. VLDL, LDL, HDL and post HDL fractions were collected for all experiments. In some experiments, HDL from individual serum samples was freshly isolated using LipiDirect HDL reagent (Polymedco, Cortland Manor, NY) following the manufacturer's protocol. Cholesterol content analysis and BCA protein assay of the supernatant containing HDL was performed and used within 48 hours after isolation.

SELDI  Sample preparation for analysis.

Lipoprotein or serum samples were prepared and processed according to the manufacturer's protocol on normal phase (NP-20), strong anion exchange (Q10), and weak cation exchange (CM 10) ProteinChip arrays (Ciphergen Biosystems, Fremont, CA). . Briefly, NP-20, Q10 and CM10 array spots were equilibrated with binding buffer (1 × PBS / 0.1% Triton X-100, pH 7) for 10 minutes at room temperature. Samples diluted with binding buffer (serum 1:25, lipoprotein fraction 1: 2) were spotted onto the array chip and incubated for 30 minutes at room temperature in a humid chamber. The chip was washed twice with binding buffer and once with HPLC H ₂ O and then air-dried. The chip was treated with cinafinic acid solution, first with 0.5 μl of 100% saturated solution and then with 1 μl of 50% saturated solution. The cinafinic acid solution is freshly prepared by saturating the EAM solution (50% acetonitrile and 0.5% trifluoroacetic acid) with cinafinic acid (3,5-dimethoxy-4-hydroxycinnamic acid).

Ciphergen Proteinchip SELDI - TOF - MS  analysis.

The array was analyzed with a Ciphergen ProteinChip Reader (Model PB SII) as described in Example 2.

Statistical analysis.

The data were analyzed using ProteinChip data analysis software version 3.2 (Ciphergen Biosystems, Fremont, CA) described in Example 2.

Identification of Proteins Showing Specific m / z Peaks

Serum protein Fractionation .

Serum was desalted on a P-6 Micro Bio-Spin chromatography column (Bio-Rad, Hercules CA) following the manufacturer's protocol. Using a Q10 anion exchange spin column (Ciphergen Biosystems, Fremont, CA), the serum was eluted with a series of buffers with decreasing pH to pH 8.5, 7.5, 7.0, 6.0, 5.0, 4.0, and 2.0 according to the manufacturer's protocol. Samples were fractionated. Protein fractions were analyzed on SELDI-TOF-MS PSII using Q10 or CM10 ProteinChip arrays. Both arrays were equilibrated with 10 mM HCl in binding buffer before use.

Serum protein purification, manual elution and SELDI - TOF - MS  Confirmation by.

Fractions identified by SELDI analysis that contained a significant majority of the peak of interest were collected and dried by centrifugal evaporation. Fractions containing the proteins were further separated on SDS-PAGE and stained with Simply Blue Safe dyes (Invitrogen, Carlsbad, Calif.). Bands with molecular weights corresponding to the peaks of interest were cut out, the gel slices were cut in half, and half of the gel slice was manually eluted as previously described (Le Bihan et al. (2004) Proteomics , 4: 2739. -2753). Briefly, the gel was dehydrated, dried in a heat block and rehydrated in an organic mixture. The gel was sonicated and then vortexed. Using the eluted protein, the presence of the peak of interest was confirmed by SELDI ProteinChip analysis. The remaining half was used for the gel digestion described below.

Trypsin digestion.

In-gel trypsin digestion was performed as previously described (Gomez et al. (2003) Mol Cell Proteomics 2: 1068-1085). Briefly, the eluate containing peaks of interest (after confirmation by SELDI) was reduced with DTT, alkylated with iodoacetamide and treated with trypsin (Promega). The gel slices were saturated with HPLC grade water to recover peptides and extracted with acetonitrile / trifluoroacetic acid. The extract was dried at low temperature SAVANT Speed Vac (Global Medical Instrumentation) and applied to μLC-MSMS as described previously (same document above). The data was used to search a mouse database using Sonar ms / ms ^™ (Genomic Solutions) and TurboSEQUEST ^™ (Thermo Electron Corp).

Electrophoresis and Immunoblot .

IEF, Tris / HCl gels and all other reagents for electrophoresis were purchased from Bio-Rad (Hercules, CA). Serum samples (2 μL) were loaded on 15% SDSPAGE, IEF (pH 3-10), Tris / HCl native (4-15%) or IEF-Tris / HCl 2D gel. For 2D gels, each lane from the IEF gel was cut according to the manufacturer's protocol (Bio-Rad) and inserted into the original gel. Serum samples were loaded onto gels according to the manufacturer's protocol (Bio-Rad) and transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ). The membranes were immunoblotted at 1: 1000 for hemoglobin (MP Biomedicals, Irvine, Calif.) Or 1: 10,000 for apoA-1 (Bethyl Laboratory, Montgomery, TX). HRP-conjugated secondary antibody (GE Healthcare) was used at a dilution of 1: 10000 and the bands were visualized with ECL detection reagent (GE Healthcare).

Hb  Spectroscopic measurement for.

FPLC fractions containing HDL were collected for the determination of Hb using a Beckman DU 640 spectrophotometer. Spectra of all samples and pure hemoglobin species were scanned from 380 to 700 nm. In order to observe the conversion of oxyhemoglobin (oxyHb) to methemoglobin (metHb), a sustained release NO donor, spermine NONOate (Cayman Chemical, Michigan) was added to the sample containing oxyHb. The "base spectrum" of a group of pure species was fitted to the measured spectrum using linear regression to deconvolve the concentrations of oxyHb and metHb (Vaughn et al . (2000) J. Biol. Chem . , 275: 2342-2348). The effectiveness of this deconvolution method was demonstrated when the total hemoglobin was converted in all samples while the consumption of oxyHb to production of metHb showed a 1: 1 ratio.

result.

m / z 14.9k and m / z 15.6k SELDI  Peaks Atheromatous formation Of Hyperlipidemia  Related to serum and HDL.

SELDI analysis using strong anion exchange (Q10) ProteinChips revealed that the peaks representing m / z 14.9k and m / z 15.6k were obtained from mouse models of four different atherosclerosis / hyperlipidemia (FIG. 20, top). Panels) and HDL (FIG. 20, bottom panel) showed at least a 7-fold increase compared to the control. All subsequent experiments for the identification and characterization of proteins exhibiting SELDI peaks of m / z 14.9k and m / z 15.6k were performed with C57BL / Serum and HDL samples obtained from 6J mice were performed.

size, pI  And TagIdent  Used, SELDI  Identification of potential candidate proteins for peaks m / z 14.9 and m / z 15.

To determine the identity of the proteins exhibiting these two SELDI peaks, we first examined the pi ranges for these two peaks. Individual serum samples obtained from C57BL / 6J mice (n = 8 per group) fed a normal or atheromatous diet for 7 days (short term) or 15 weeks (long term) were subjected to anion exchange fractionation as described below. It was. Fractions were eluted with buffers of different pH and further analyzed on CM10 and Q10 SELDI chips (FIG. 21). Most of the intensities representing the two peaks m / z 14.9k and m / z 15.6k were eluted with buffers ranging from pH 7.5 to pH 8.0 (FIG. 21). On-line TagIdent (protein database) searches were performed using the size determined by SELDI-TOF-MS analysis and the corresponding pI determined by anion exchange fractionation (FIG. 21). Using search conditions that allowed 0.5% size error and a + 2 pI range, Hb-alpha and Hb-beta were identified as potential candidate proteins for 14.9 kDa and 15.6 kDa, respectively. Based on the literature, the pI range for free Hb is 7.5-8.5. Interestingly, these two peaks, representing m / z 14.9k and m / z 15.6k, were also shown to be related to fractions from atheromatous serum eluted with pH 7.0 and pH 4.0 buffers (FIG. 21). These data suggested that i) Hb in atheromatous samples had different chemical properties, or ii) proteins representing m / z 14.9k and m / z 15.6k were different from Hb. As shown below, by the following studies, these peaks actually showed Hb, and the different physical and chemical properties of the HDL-associated Hb resulted from a close association with other HDL-associated proteins, such as haptoglobin. Appeared to be.

Fractionation  And peptides by trypsin Sectioning  And Tandem  By the analysis by mass spectrometer, the above two Biomarker  The identity of protein Hb Alpha and Hb -Confirmed to be beta.

To further identify the identity of the two biomarkers, peaks corresponding to their size were partially purified from mouse serum after dealbuminization and anion exchange chromatography. After trypsin digesting the partially purified proteins, μLC-MSMS analysis and the resulting fragments were retrieved from the human protein databases (Snar and SEQUEST). As a result, 14.9 kDa protein was identified as alpha-Hb and 15.6 kDa protein was identified as beta-Hb.

Progenitor-inflammatory in mouse model HDL  Potential for As marker  Identification of hemoglobin.

To further demonstrate the presence of Hb in atheromatous serum and HDL by a non-SELDI method, we first tested serum samples from mice on an oncogenic diet on SDS-PAGE. Western blotting against Hb. The total amount of Hb in serum samples did not differ significantly between normal and atheromatous serum (FIG. 22A).

It should be noted that the total concentration of Hb in the serum (ie non-RBC Hb) is about 10 μΜ. In contrast, the concentration of Hb in whole blood is greater than 1 M. As such, only about 0.001% of Hb is found outside of RBC in the blood. As shown in FIG. 22A, the amount of non-RBC Hb in serum is not different in mice fed a normal diet compared to mice fed an atheromatous diet.

However, in FPLC fractionated lipoproteins of normal serum (ie, serum from mice fed a normal diet), Hb is associated with later HDL (pHDL) fractions, while Hb in atheromatous serum is associated with the HDL fraction. Were associated (FIG. 22B). Furthermore, the heme content of Hb obtained by OD measurements on the individual FPLC fractions at 410 nm confirmed the observation in FIG. 22B (FIG. 22C). In mice fed with an atheromatous diet for 15 weeks, Hb was found exclusively in HDL fractions (FIG. 22B). These experiments suggest that Hb is a marker for pro-inflammatory HDL in mice.

Atheromatous formation  Hemoglobin associated with serum exhibits unique physical and chemical properties.

Western blot analysis of serum samples showed no significant difference in Hb mass between normal and atheromatous serum (FIG. 22A), whereas the association of Hb to HDL at the time of feeding the atheromatous diet changed. It suggests that the hemoglobin mass remained unchanged. Experiments for the discovery of biomarkers associated with atheromatous sera were performed on SELDI using a Q10 array, which selects proteins with pI <7. Furthermore, in the anion exchange fractionation experiment described above, a significant amount of the two peaks representing m / z 14.9k and m / z 15.6k is associated with atheromatous serum fractions eluted by pH 7.0 and pH 4.0 buffers. (FIG. 21). These data indicate that i) the mass of Hb does not change significantly under atherogenic conditions, ii) the chemical properties of Hb change under atherogenic conditions as evidenced by the unique pI values (FIG. 21), and iii) atherogenicity. As a result of changes in HDL and / or changes in Hb under sexual conditions, it is suggested that Hb associates with HDL, after 15 weeks of atheromatous diet, Hb was associated only with HDL.

To characterize the unique properties of Hb in atheromatous serum, SELDI analysis was performed on FPLC lipoprotein fractions and serum samples from long-term studies (W15) using NP20 and Q10 arrays. NP20 arrays that bind all proteins captured the same amount of Hb from both normal and atheromatous serum samples (FIG. 23A, left panel). Furthermore, Hb peaks on NP-20 arrays were associated with pHDL fractions in non-atherogenic samples, while totally associated with HDL fractions in atheromatous samples (FIG. 23A, left panel). These data are consistent with Western blotting analysis after SDS-PAGE shown in FIG. 22B. On the other hand, the Q10 array captured Hb in serum and HDL fractions obtained only from atheromatous samples (FIG. 23A, right panel), indicating that the associated Hb in atheromatous serum has unique properties and is associated with HDL. Suggests that. To further investigate the development of Hb with unique pi in an atheromatous situation, the SELDI-proven serum fractions of the anion exchange columns of FIG. 21 were analyzed by SDS-PAGE followed by Western analysis. The atheromatous serum contained Hb in anion exchange column fractions from pH7 and pH4 (FIG. 23B).

HDL  Associated with fractions Hb of  Feature Analysis

To further identify the physicochemical properties of Hb associated with HDL, serum samples from the D7 and W15 groups and HDL fractions from the D7 group were subjected to iso-electric focusing (IEF) and native gel electrophoresis. It was applied to Youngdong. The IEF gel demonstrated that normal Hb had a pI of about 7.5 while Hb in atheromatous serum had a reduced pi of about 4 (FIG. 24). The transition from normal Hb (pI 7.5) to modified Hb (pI 4.0) was evident in samples from the D7 group (FIG. 24). These changes were not due to changes in RBCs because RBCs isolated from the same group of mice showed Hb with normal pi (FIG. 24). Furthermore, the Hb associated with the HDL fraction from the D7 group showed a modified version of Hb (FIG. 24). The original gels showed an association of Hb immunoreactivity with high molecular weight (HMW) particles in atheromatous serum samples, which were exclusively associated with HDL after 15 weeks of atheromatous diet (FIG. 25). Furthermore, the IEF / original 2D gel (FIG. 12) confirmed that Hb from atheromatous serum had a number of morphologies (based on pi), which primarily associate with HMW particles. In order to determine the specific form of Hb associated with HDL (oxyHb or metHb), the mixture fractions of HDL were spectroscopically analyzed. The main form of Hb associated with HDL was oxyHb and metHb was found to be small (Figure 26 and Table 15).

discussion

Protein profiling is an efficient way of determining differentially expressed and / or associated proteins in serum samples, allowing for the rapid evaluation of biologically important functions. Ciphergen Biosystems (Fremont, CA) has developed ProteinChip technology combined with surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS) that facilitates protein profiling of complex biological mixtures (Rubin). And Merchant (2000) Am. Clin. Lab. 19: 28-29; Weinberger et al. (2002) Curr. Opin . Chem . Biol . 6: 86-91; Fung et al . (2001) Curr . Opin . Biotechnol . 12:65 -69; Issaq et al . (2002) Biochem . Biophys . Res . Commun . 292: 587-592). SELDI is unique in its ability to analyze trace (low femtomol) analytes from complex sources (serum, urine, feces, CSF, tissue culture extracts, cell lysates). "Preactivated" surface ProteinChip arrays also have "bait" molecules (e.g., charge, hydrophobicity, specific binding affinity, antibodies) covalently bound to the chip surface when the target analyte of interest is known, such as various different choices. It functions as an open platform for the figure. The efficacy of SELDI-TOF-MS technology for the discovery of cancer protein markers in serum has recently been demonstrated (Wright et al. (1999) Prostate Cancer Prostatic Dis . 2: 264-276; Li et al . (2002) Clin . Chem . 48: 1296-1304). We have successfully used the SELDI-TOF-MS system and have previously reported the identification of biomarkers for early detection of ovarian cancer (Kozak et al. (2003). Proc . Natl . Acad . Sci . , USA, 100: 12343-12348; Kozak et al. (2005) Proteomics 5: 45 89-96).

By database search after pI measurements of two peaks representing m / z 14.9k and m / z 15.6k (FIG. 21), Hb-alpha and Hb-beta are likely associated with pro-inflammatory HDL in atheromatous serum It was found to be a biomarker. These findings were confirmed using μLC-MSMS methods. The inventors were initially concerned with the finding because mechanical handling and / or sample preparation is likely to cause RBC dissolution and Hb release. However, these experiments were then carefully repeated to conclude that Hb is not an artificial product but rather a specific and significant marker for pro-inflammatory HDL present in atheromatous serum. First, we showed no difference in total Hb mass between serum samples obtained from mice fed a normal and atheromatous diet (FIG. 22B). The total concentration of Hb (ie non-RBC Hb) in the serum is approximately 10 μΜ. In contrast, the concentration of Hb in whole blood is greater than 1 M. That is, only about 0.001% of Hb is found outside of RBC in the blood. As shown in FIG. 22A, the amount of non-RBC Hb in serum is not different in mice fed a normal diet compared to mice fed an atheromatous diet.

Secondly, during the conversion of normal HDL to pro-inflammatory HDL (D7 normal versus D7 atheromatous diet), we found no modification in Hb (quantity and quality) in the RBC lysate obtained from the same mouse. This suggests once again that the Hb association for pro-inflammatory HDL is a specific phenomenon under pro-inflammatory situations. Third, the association of Hb to pro-inflammatory HDL is evident by the comparison of Hb in D7 and W15 lipoprotein samples (FIGS. 22B and 24 and 25), depending on the degree of pro-inflammatory situation. Finally, in four different atherosclerotic / hyperlipidemic models, including apoE deficient mice with pro-inflammatory HDL on a normal diet, Hb was found associated with HDL. When combined, the results indicate that HDL-associated Hb is a marker for pro-inflammatory HDL.

Normal pi for Hb has been reported to be pi 7.0 to 8.0. We found that Hb associated with pro-inflammatory HDL has at least two Hb species with distinct pI values (FIGS. 21 and 24). After 15 weeks of atheromatous diet, all Hb associated with HDL actually expressed this abnormal pI. The pI change in Hb after the atheromatous diet was shown not to be due to a change in hemoglobin, but rather to a protein with which hemoglobin was closely associated in HDL. Since most of the Hb under these circumstances has been shown to be oxyhemoglobin, the oxidative stress induced by atheromatous diets is likely responsible for these changes. The absence of this change in RBC Hb from the same mouse suggests that under these circumstances free Hb may have been differentially affected. The fact that there was no difference in total serum Hb in normal or atheromatous diets suggests that increased RBC lysis was not a factor in this process.

Haptoglobin (Hp) and hemopexin (Hx) are hemoglobin (Hb) (K), respectively._d 1 pM) and heme (K_d Plasma protein with the largest binding affinity for <1 pM). They are primarily expressed in the liver and belong to a family of acute phase proteins in which synthesis is induced during the inflammatory process (Bowman and Kurosky (1982)Adv Hum Genet . 12: 189-261; Altruda et al. (1985)Nucleic Acids Res . 13: 3841-3859). It is established that Hb (one of the most abundant and functionally important protein in red blood cells) released from red blood cells is highly toxic due to the oxidative properties of heme, which participate in the Fenton reaction and produce free radicals that cause cellular damage (Hoffman). Etc (1995)Hematology : Basic Principle and Practice . Second Edition New York, NY: Churchill Livingstone. Toxicity of heme is increased by heme hydrophobicity, which makes it possible to penetrate into lipid membranes and other lipophilic compartments when heme is not associated with proteins (Balla et al. (1993)Proc. Natl . Acad . Sci ., USA, 90: 9285-9289). Usually, a small amount of extravascular hemolysis occurs during enucleation of erythrocytes and destruction of senescent red blood cells, thereby causing Hb release into the plasma. Under pathological conditions associated with bleeding, hemochromatosis, ischemic reperfusion, or intravascular hemolysis such as malaria, large amounts of free Hb are released (Wagener et al. (2001)Trends Pharmacol Sci . 22: 52-54). Once in plasma, free Hb dissociates rapidly into dimers bound by Hp. Metabolism of plasma Hb is considered a major function of tissue macrophages, which absorb Hb-Hp complexes through the macrophage scavenger receptor CD1 63 (Schaer et al. (2006)Blood 107: 373-380; Fabriek et al. (2005)Immunobiology 210: 153-160), which can be incorporated into cells (Kristiansen et al. (2001)Nature 409: 198-201). Interestingly, very recent studies have identified low density lipoprotein receptor-associated protein (LRP) / CD9 1 as a receptor responsible for scavenging hemopexin-hem complexes (Hvidberg et al. (2005)Blood 106: 2572-2579). LRP / CD9 1 is expressed in many cell types, including macrophages and hepatocytes, which can incorporate the heme-Hx complex into cells via receptor-mediated endocytosis (Hunt et al. (1996)).J Cell Physiol . 168: 71-80). In the experiments reported here, we did not see the release of Hb from RBC, but rather only the conversion of existing Hb into a form associated with HDL fractions (not previously reported). Under oxidative stress conditions, Hb-Hp-Hx complexes are formed, and they are likely associated with HDL to be quickly removed from the circulation. Indeed, Hp has been reported to associate with apoA1, a major protein component of HDL (Rademacher et al. (1987).Anal Biochem . 160: 119-126; Kunitake et al. (1994)Biochemistry 33: 1988-1993; Porta et al. (1999)Zygote 7: 67-77; Spagnuolo et al. (2005)J. Biol . Chem .. 280: 1193-1198). Hp association of apoA1 alters HDL function (Balestrieri et al. (2001)Mol Reprod Dev . 59: 186-191; Cigliano et al. (2001)Steroids 66: 889-896). The data in FIGS. 12-15 suggest that non-RBC hemoglobin, haptoglobin, and hemopexin are all present in the same complex in HDL under atheromatous conditions.

Hb is a known marker for blood sugar, oxidative stress, hypertension, insulin resistance, obesity, and injuries and diseases associated with diabetes (Zhang et al. (2004) Proteomics 4: 244-256; de Valk and Marx (1999) Arch. Intern Med . 159: 1542-1548; Alayash et al. (2001) Antioxid Redox Signal 3: 313-327). Hb is also considered toxic because free Hb is also a potential oxidant due to its heme (Fe) and heme-bonded reactive radicals (Alayash (1999) Nat Biotechnol . 17: 545-549), these radicals have also been shown to oxidize LDL in vivo (Paganga et al. (1992) FEBS Lett . 303: 154-158; Miller et al. (1996) Arch Biochem Biophys . 326: 252-260; Ziouzenkova et al. (1999) J. Biol . Chem . . 274: 18916-18924). Our findings show for the first time that Hb associates with HDL fractions under oxidative stress in mice. Pro-inflammatory HDL in atheromatous serum contains lipid hydroperoxide (LOOH), lacks paraoxonase activity, activates monocytes, does not prevent oxidation of LDL, and exhibits less cholesterol efflux. We report here that Hb specifically associates with pro-inflammatory HDL in atheromatous mice. Without being bound by a particular theory, the inventors believe that the Hb association for HDL is implicated in the conversion of HDL anti-inflammatory to pro-inflammatory.

In conclusion, in animal models of atherosclerosis, Hb associates with pro-inflammatory HDL. Expanding to humans, the inventors believe that HDL-associated Hb can function as a marker for pro-inflammatory HDL.

Example  4

Qiagen  Albumin removal Column  Used LDL  Protocol of Coagulation Assay

material.

Typical materials for LDL aggregation assays using albumin removal columns are shown in Table 16.

Material of LDL Coagulation Assay material Where To Buy / Supply Source Catalog number 1 2 3 4 5 Qproteome albumin / IgG removal column LDL 50 mM Tris, 150 mM NaCl, 2 mM CaCl ₂ buffer 96 well plates Phospholipase C from Bacillus cereus (PLC) Qiagen Ultracentrifuge Not applicable Costar Sigma 37521 n / a 3596 P6621

Way:

Preparation of the Samples.

1. Dilute 25 μl of serum or plasma with 75 μl dilution buffer.

2. Albumin / IgG Removal The spin column is briefly centrifuged at 500 x g to remove resin from the screw cap.

3. Remove the screw cap, break the bottom partition of the spin column, and drain the storage buffer using gravity flow.

4. Equilibrate the spin column by pipetting 2 x 0.5 mL aliquots of dilution buffer and draining each by gravity flow.

5. Close the spin column with the cap for the QIAfilter Cartridge.

6. Apply the sample prepared in step 1 onto the column.

7. Close the lid of the spin column and shake vigorously to obtain a uniform suspension. It is incubated for 5 minutes on a shaker at room temperature.

8. Remove the QIAfilter Cartridge and transfer the spin column to a clean centrifuge tube.

9. Loosen the column cap 1/4 turn.

10. Collect flow-through by centrifugation at 500 x g for 10 seconds.

11. Wash the column with 2 × 100 μL aliquots of dilution buffer and collect each wash fraction by centrifugation at 500 × g for 10 seconds.

12. Combine the flow-through fraction from step 10 and the two wash fractions from step 11.

Apo  Eliminate B-containing protein and measure cholesterol

1. ApoB containing protein was removed from the albumin removal sample using dextran sulfate precipitation. In precipitation with dextran sulfate, Sigma HDL cholesterol reagent containing dextran sulfate and magnesium ions was dissolved in distilled water. 50 microliters of dextran sulfate (1.0 mg / ml) was mixed with 500 uL of each sample and incubated at room temperature for 5 minutes and then centrifuged at 3,000 g for 10 minutes. Supernatants containing HDL were used for this experiment.

2. Measure total cholesterol in albumin / apoB depleted HDL supernatant using standard cholesterol assay.

3. Add HDL supernatant to each well at a concentration of 10 μg.

Phospholipase C ( PLC Manufacture of

1. Add enough ddH ₂ O to the vial (250 units / vial) of the PLC to make 56.3 units / mL. For example, 4.4 mL ddH ₂ O is added to a vial containing 250 units.

2. Short vortexing.

3. Aliquot 200 μl of PLC solution into the Eppendorf tube.

Freeze at -20 ° C.

LDL  + Of sample Incubation

1. Add to the wells (all 3 controls, samples, etc.):

Ingredients of Wells sample Buffer (μl) LDL (μl) sample LDL only LDL + PLC LDL + PLC + Sample 190 170 130 10 10 10   40 Note: Each well should have 75 μg LDL (eg, if the concentration of LDL is 8.6 mg / mL, add 8.7 μl). The values may vary depending on the LDL and the concentration of the sample.

2. Incubate the plate on a nutator at 37 ° C. for 1 hour.

3. Read the plate at a wavelength of 478 nm on a plate reader.

PLC  inclusive Incubation

1. Add 20 μL of PLC solution to each well except “LDL only”.

2. Add 200 μL of buffer to the 200 μL of PLC solution.

3. Short vortexing.

4. Add 20 μl of the diluted PLC solution to each well as indicated.

5. Read plates at 478 nm at 0, 5, 10, 30, 45, and 60 minutes.

Example  5

D-4F treatment in mice was associated with hemoglobin and its Scavenger  High density To lipoproteins  Reduce association.

purpose.

We previously reported that D-4F, an apolipoprotein A-I (apoA-I) mimetic in mice and monkeys, converted HDL from pro-inflammatory to anti-inflammatory. We found that in animal models of atherosclerosis, hemoglobin (Hb) associates with HDL and contributes to the pro-inflammatory properties of HDL under atheromatous conditions, suggesting that D-4F treatment in animal models of atherosclerosis It was intended to determine if it could reduce the association of Hb to pro-inflammatory HDL.

Method and result.

The data in FIG. 2 show that HDL taken from apoE deficient mice (mouse model of atherosclerosis) is pro-inflammatory. After administration of the oral apoA-I mimetic peptide (D-4F), the HDL was converted to anti-inflammatory. As shown in Figures 3 and 4, with changes in the inflammatory properties of HDL, the concentrations of hemopexin and haptoglobin in the serum of the mice decreased.

Assays for HDL from the mouse indicated that the haptoglobin and hemopexin content in the mouse HDL was parallel to the conversion from pro-inflammatory HDL to anti-inflammatory and without change in the content of apoA-I in the HDL. Was found to decrease (FIG. 5). With decreasing levels of serum and HDL hemopexin and haptoglobin, increased concentrations of non-RBC hemoglobin migrated along characteristics similar to normal RBC hemoglobin (Figure 6) and decreased hemoglobin content of HDL (Figure 7, Man There is a bar graph on the right). As shown in FIGS. 29 and 30, D-4F administration also significantly reduced the contents of transferrin and myeloperoxidase in HDL-supernatant.

conclusion.

Hb and its scavenger proteins hemopexin and haptoglobin are components of pro-inflammatory HDL. One mechanism by which D-4F converts pro-inflammatory HDL to anti-inflammatory HDL can be by preventing and / or reversing the association of oxidation-promoting Hb containing proteins with HDL. Treatment with pro-inflammatory HDLs with agents that convert to anti-inflammatory HDLs clearly involved changes in HDL-associated hemoglobin, haptoglobin hemopexin, transferrin, and myeloperoxidase. These results indicate that the assays described herein will be useful in monitoring therapies that improve dysfunctional HDL and weaken atherosclerosis.

It is apparent that the embodiments and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art in view of them and they will be included within the spirit and scope of the present application and the scope of the appended claims. Do. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (38)

A method of detecting or quantifying pro-inflammatory HDL in a mammal, comprising: Providing a sample comprising HDL as a biological sample from said mammal; And Detecting at least two proteins associated with HDL, wherein the at least two proteins are proteins having an m / z ratio of about 9.3, a protein having an m / z ratio of about 14.9, a protein having an m / z ratio of about 15.6, m the protein having an / z ratio of about 15.8, a protein having an m / z ratio of about 16.2, a protein having an m / z ratio of about 16.5, a protein having an m / z ratio of about 18.6, and a protein having an m / z ratio of about 19.5, and , Detection of the two or more proteins associated with HDL indicates that the HDL is pro-inflammatory HDL. The method of claim 1, wherein said detecting comprises detecting at least three of said proteins. The method of claim 1, wherein said detecting comprises detecting at least four of said proteins. The method of claim 1, wherein said detecting comprises detecting at least five of said proteins. The method of claim 1, wherein said detecting comprises detecting at least six of said proteins. The method of claim 1, wherein said detecting comprises detecting at least seven of said proteins. The method of claim 1, wherein said detecting comprises detecting all eight of said proteins. The method of claim 1, wherein the mammal is a human. The method of claim 1, wherein the mammal is a human diagnosed with atherosclerosis. The method of claim 1, wherein said mammal is a human diagnosed with a risk of atherosclerosis. The method of claim 1, wherein said detecting comprises immunoassay. A method for detecting or quantifying pro-inflammatory HDL in a mammal, comprising: measuring the level of heme-containing and / or heme-binding protein (s) associated with HDL from said mammal, wherein An increase in the level of heme-containing and / or heme-binding protein (s) compared to the level of heme-containing and / or heme-binding protein found in normal anti-inflammatory HDL is that the HDL is a pro-inflammatory HDL. Indicating that. The method of claim 12, wherein the heme-containing and / or heme-binding protein comprises one or more proteins selected from the group consisting of hemoglobin, haptoglobin, hemopexin, transferrin, soluble CD163, and myeloperoxidase. . 13. The method of claim 12 comprising measuring the amount of hemoglobin associated with HDL and measuring the amount of haptoglobin associated with HDL. 15. The method of claim 14 comprising calculating the products of hemoglobin and haptoglobin associated with HDL. 13. The method of claim 12, further comprising determining the level of said same heme-containing and / or heme-binding proteins in the non-lipoprotein fraction of plasma, wherein heme-containing and / or in HDL Or an increase in the ratio of heme-containing and / or heme-binding protein in the non-lipoprotein fraction of heme-binding protein to plasma is increased compared to the ratio found in subjects with normal anti-inflammatory HDL. A method indicating that HDL from an animal is pro-inflammatory HDL. 13. The method of claim 12, wherein said measuring comprises immunoassay. 13. The method of claim 12, wherein said measuring comprises an ELISA. A method of detecting or quantifying pro-inflammatory HDL in a mammal, comprising: Measuring the level of one or more heme-containing and / or heme-binding protein (s) associated with HDL from said mammal; Measuring the level of one or more of said same heme-containing and / or heme-binding protein (s) in plasma from said mammal, comprising: HDL heme-containing and / or heme-binding protein to plasma heme-containing and / or Or the ratio of heme-binding protein greater than 1 indicates that the HDL from the mammal is pro-inflammatory HDL. The method of claim 19, wherein the heme-containing and / or heme-binding protein comprises one or more proteins selected from the group consisting of hemoglobin, haptoglobin, hemopexin, transferrin, soluble CD163, and myeloperoxidase. . 20. The method of claim 19, wherein the ratio of HDL heme-containing and / or heme-binding protein to plasma heme-containing and / or heme-binding protein in at least two of said proteins is greater than or equal to HDL from said mammal. A method of indicating pro-inflammatory HDL. 20. The method of claim 19 wherein the ratio of HDL heme-containing and / or heme-binding protein to plasma heme-containing and / or heme-binding protein in at least three of said proteins is greater than or equal to HDL from said mammal. A method of indicating pro-inflammatory HDL. The method of claim 19, wherein the ratio of HDL heme-containing and / or heme-binding protein to plasma heme-containing and / or heme-binding protein in one, two, three or four of said proteins is 1, 0.9. Exceeding a value selected from the group consisting of, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 indicates that the HDL from the mammal is pro-inflammatory HDL. A method of detecting or quantifying pro-inflammatory HDL in a mammal, comprising: Determining the level of heme associated with HDL from the mammal, wherein the level of heme associated with HDL is increased relative to the level of heme associated with protective HDL. Indicating that it is. The method of claim 24, wherein said increased level is statistically significant at a confidence level of at least 90%, 95%, 98%, or 99%. A method of detecting or quantifying pro-inflammatory HDL in a mammal, comprising: Determining the iron content of the HDL from the mammal, wherein the level of iron content of the HDL is increased compared to the iron content of normal anti-inflammatory HDL, wherein the HDL from the mammal is a pro-inflammatory HDL Indicating that. 27. The method of claim 26, wherein said increased level is statistically significant at a confidence level of at least 90%, 95%, 98% or 99%. A method of detecting or quantifying pro-inflammatory HDL in a mammal, comprising: Determining the level of iron-containing proteins associated with HDL from the mammal, wherein the level of iron-containing proteins associated with HDL is associated with the level of iron-containing proteins associated with normal anti-inflammatory HDL. Increased compared to indicate that the HDL from the mammal is pro-inflammatory HDL. The method of claim 28, wherein said increased level is statistically significant at a confidence level of at least 90%, 95%, 98%, or 99%. A method of detecting or quantifying pro-inflammatory HDL, comprising measuring the nitrogen monoxide consumption capacity of the HDL, wherein the increase in the nitrogen monoxide consumption capacity of the HDL compared to the nitrogen monoxide consumption capacity of the normal anti-inflammatory HLD. A method that indicates the presence, amount or activity of pro-inflammatory HDL. 31. The method of claim 30, wherein said nitric oxide is chemically produced. 31. The method of claim 30, wherein said nitric oxide is measured by an electronic signal. 33. A method of assaying for the presence or predisposition of a pathology characterized by an inflammatory response in a mammal, comprising performing any one or more of the assays of claims 1 to 32, wherein the positive test result is Method that is a marker for presence or predisposition. 34. The method of claim 33, wherein the pathology is atherosclerosis, stroke, leprosy, tuberculosis, systemic lupus erythematosus, rheumatic polymyalgia, nodular polyarteritis, scleroderma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, Alzheimer's disease, chronic Renal failure, diabetes, diabetic retinopathy, diabetic kidney disease, transplant rejection, graft atherosclerosis, reperfusion ischemia, adult respiratory syndrome, congestive heart failure, glomerulitis, metabolic syndrome, multiple sclerosis, sepsis syndrome, sickle cell disease, Vascular dementia, Crohn's disease, endothelial dysfunction, arterial dysfunction, AIDS, rheumatoid polymyalgia, nodular polyarteritis, scleroderma, idiopathic pulmonary fibrosis, coronary artery calcification, calcified aortic stenosis, osteoporosis, bacterial infection, viral infection, fungal infection , Autoimmune disorders, and rheumatoid arthritis. A method of treating a human with a pathology characterized by an inflammatory response, comprising: Performing any one or more of the assays of claims 1 to 32; And Prescribing more aggressive treatment for subjects with positive test results in the assays. A method of detecting or quantifying pro-inflammatory HDL in a mammal, the method comprising contacting HDL from a mammal with LDL and measuring aggregation of the LDL, wherein the level of LDL aggregation is normal anti- An increase in comparison to the aggregation of LDL in contact with inflammatory HDL indicates that the HDL from the mammal is a pro-inflammatory HDL. The method of claim 36, wherein said aggregation is determined by spectroscopic measurement of LDL aggregation rate. 37. The method of claim 36, wherein said aggregation is determined using an albumin depletion column.

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